Cyanine dyes

ABSTRACT

The invention provides a novel class of cyanine dyes that are functionalized with sulfonic acid groups and a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/489,143 filed Sep. 17, 2014, which is a continuation of U.S.application Ser. No. 13/218,428 filed Aug. 25, 2011, which claimspriority to Provisional Application Nos. 61/377,004, filed on Aug. 25,2010, 61/377,022, filed on Aug. 25, 2010, 61/377,031, filed on Aug. 25,2010, 61/377,038, filed on Aug. 25, 2010, and 61/377,048, filed on Aug.25, 2010 the disclosures of which are incorporated herein by referencein their entirety for all purposes.

FIELD OF INVENTION

The present invention relates generally to the synthesis of fluorescentcompounds that are analogues of cyanine dyes. The compounds of theinvention are fluorophores that are derivatized to allow their facileattachment to another moiety. The invention also relates to improvedmethods for sequencing and genotyping nucleic acid in a single moleculeconfiguration. An exemplary method involves detection of singlemolecules of fluorescent labels released from a nucleic acid duringsynthesis of an oligonucleotide.

BACKGROUND

There is a continuous and expanding need for rapid, highly specificmethods of detecting and quantifying chemical, biochemical andbiological substances as analytes in research and diagnostic mixtures.Of particular value are methods for measuring small quantities ofnucleic acids, peptides, saccharides, pharmaceuticals, metabolites,microorganisms and other materials of diagnostic value. Examples of suchmaterials include narcotics and poisons, drugs administered fortherapeutic purposes, hormones, pathogenic microorganisms and viruses,peptides, e.g., antibodies and enzymes, and nucleic acids, particularlythose implicated in disease states.

The presence of a particular analyte can often be determined by bindingmethods that exploit the high degree of specificity, which characterizesmany biochemical and biological systems. Frequently used methods arebased on, for example, antigen-antibody systems, nucleic acidhybridization techniques, and protein-ligand systems. In these methods,the existence of a complex of diagnostic value is typically indicated bythe presence or absence of an observable “label” which is attached toone or more of the interacting materials. The specific labeling methodchosen often dictates the usefulness and versatility of a particularsystem for detecting an analyte of interest. Preferred labels areinexpensive, safe, and capable of being attached efficiently to a widevariety of chemical, biochemical, and biological materials withoutsignificantly altering the important binding characteristics of thosematerials. The label should give a highly characteristic signal, andshould be rarely, and preferably never, found in nature. The labelshould be stable and detectable in aqueous systems over periods of timeranging up to months. Detection of the label is preferably rapid,sensitive, and reproducible without the need for expensive, specializedfacilities or the need for special precautions to protect personnel.Quantification of the label is preferably relatively independent ofvariables such as temperature and the composition of the mixture to beassayed.

A wide variety of labels have been developed, each with particularadvantages and disadvantages. For example, radioactive labels are quiteversatile, and can be detected at very low concentrations. However, suchlabels are expensive, hazardous, and their use requires sophisticatedequipment and trained personnel. Thus, there is wide interest innon-radioactive labels, particularly in labels that are observable byspectrophotometric, spin resonance, and luminescence techniques, andreactive materials, such as enzymes that produce such molecules.

Labels that are detectable using fluorescence spectroscopy are ofparticular interest because of the large number of such labels that areknown in the art. Moreover, as discussed below, the literature isreplete with syntheses of fluorescent labels that are derivatized toallow their attachment to other molecules, and many such fluorescentlabels are commercially available.

Fluorescent nucleic acid probes are important tools for geneticanalysis, in both genomic research and development, and in clinicalmedicine. As information from the Human Genome Project accumulates, thelevel of genetic interrogation mediated by fluorescent probes willexpand enormously. One particularly useful class of fluorescent probesincludes self-quenching probes, also known as fluorescence energytransfer probes, or FET probes. The design of different probes usingthis motif may vary in detail. In an exemplary FET probe, both afluorophore and a quencher are tethered to a nucleic acid. The probe isconfigured such that the fluorophore is proximate to the quencher andthe probe produces a signal only as a result of its hybridization to anintended target. Despite the limited availability of FET probes,techniques incorporating their use are rapidly displacing alternativemethods.

To enable the coupling of a fluorescent label with a group ofcomplementary reactivity on a carrier molecule, a reactive derivative ofthe fluorophore is prepared. For example, Reedy et al. (U.S. Pat. No.6,331,632) describe cyanine dyes that are functionalized at anendocyclic nitrogen of a heteroaryl moiety with hydrocarbon linkerterminating in a hydroxyl moiety. The hydroxyl moiety is converted tothe corresponding phosphoramidite, providing a reagent for conjugatingthe cyanine dye to a nucleic acid. Waggoner (U.S. Pat. No. 5,627,027)has prepared derivatives of cyanine and related dyes that include areactive functional group through which the dye is conjugated to anotherspecies. The compounds set forth in Ohno et al. (U.S. Pat. No.5,106,990) include cyanine dyes that have a C₁-C₅ hydrocarbyl linkerterminated with a sulfonic acid, a carboxyl or a hydroxyl group. Randallet al. (U.S. Pat. Nos. 6,197,956; 6,114,350; 6,224,644; and 6,437,141)disclose cyanine dyes with a linker arm appended to an endocyclicheteroaryl nitrogen atom. The linkers include a thiol, amine or hydroxylgroup, or a protected analogue of these residues. Additional linkerarm-cyanine dyes are disclosed by Brush et al. (U.S. Pat. Nos.5,808,044; 5,986,086). These cyanine dyes are derivatized at bothendocyclic heteroaryl nitrogen atoms with a hydrocarbyl linkerterminating in a hydroxyl moiety. One hydroxyl moiety is converted tothe corresponding phoshporamidite and the other is protected as adimethoxytrityl ether.

Cyanine dyes are particularly popular fluorophores and are widely usedin many biological applications due to their high quantum yield and highmolar absorbtivity. Cyanine dyes are, however, susceptible tophotobleaching during prolonged excitation. Moreover, due the rigidplanar structure of these compounds, they have a tendency to stack andself-quench. Thus, provision of cyanine dyes having an enhancedbrightness and decreased tendency to stack, thereby mitigating theeffects of photobleaching and stacking is an important object.Furthermore, cyanine dyes that are hydrophilic are less attracted toother species such as proteins and surfaces, which reduces adventitiousbinding of the fluorophore and enhances the precision and accuracy ofassays and other analyses utilizing cyanine fluorophores. The presentinvention meets these objects and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a class of cyanine-based fluorophoresmodified to improve their fluorescent and other physicochemicalproperties. Thus, it is a general object of the invention to providecyanine dyes that are hydrophilic, are resistant to photobleaching, ormaintain a high level of brightness despite photobleaching, and have alower tendency to stack or otherwise aggregate than current cyaninefluorophores.

Exemplary dyes of the invention find particular use in DNA sequencingmodalities, particularly single molecule sequencing modalities. Previousdyes used in such applications have had less than ideal properties. Forexample, certain dyes give suboptimal performance, because, as wasdiscovered, the dyes are insufficiently hydrophilic, insufficientlybright, do not emit steadily (i.e., blink), undergo photobleaching uponprolonged irradiation or they aggregate. These deficiencies can causemisreads in DNA sequencing analyses, providing inaccurate results. Invarious embodiments, the present invention provides a solution to one ormore of these factors contributing to suboptimal dye performance. Invarious embodiments, the hydrophilicity of the dyes is enhanced by theaddition to the cyanine core or a side group attached to the cyaninecore of a water-soluble polymer, sulfonic acid, or carboxylic acidmoieties or groups containing sulfonic acid or carboxylic acid moieties.Moreover, it was discovered that substitution of a cyanine dye withcharged, hydrophilic moieties protects the cyanine chromophore from thedye's microenvironment and reduces blinking, aggregation andphotobleaching. Thus, in various embodiments, the dyes are brighter,more photostable and their emission is more constant. Furthermore, forDNA sequencing, particularly single molecule sequencing, resolution ofthe absorbance of the dye emissions is important to sensitivity andaccuracy of the measurements underlying the sequence determination.Accordingly, in various embodiments, the present invention provides dyeswith emissions tuned to achieve useful levels of resolution in theemission peaks of the dyes when they are used in combinations of 2, 3, 4or more different dyes attached to nucleic acids. Thus, in variousembodiments, the present invention provides a solution to the problem.In exemplary embodiments, the dyes of the invention provide at least a2%, at least a 5%, at least a 7% or at least a 10% improvement inreadlength in a single molecule DNA sequencing protocol when comparedwith dyes that are not functionalized as are the dyes of the invention.

In exemplary embodiments, the dyes of the invention are utilized in DNAsequencing in real time using a single polymerase enzyme attached to thebottom of the small nano-meter size hole called zero-mode waveguide(ZMW). Fluorescent signals of 4 different colors that correspond to 4different DNA bases: A, G, C, T are detected. Since the most robustmethodologies read through as many bases on a template oligonucleotideas possible, it is desirable to utilize dyes that do not limit thereadlength or the accuracy of the measurements. The water-soluble,cyanine dyes of the invention are of use in such measurements and insome embodiments increase the accuracy of the measurements by at least2%, at least 5%, at least 7% or at least 10% in a single molecule DNAsequencing protocol when compared with dyes that are not functionalizedas are the dyes of the invention.

In an exemplary embodiment, the invention provides cyanine dyesderivatized with multiple ionizable groups such as sulfonic orcarboxylic acids. Exemplary fluorophores of the invention also includewithin their structure(s) a versatile linker arm, the structure andposition of which is readily alterable, thereby allowing the conjugationof the label through a variety of positions on the cyanine nucleus to acarrier molecule. The cyanine-based labels are readily attached to alabel, such as a nucleic acid, using techniques well known in the art,or modifications of such techniques that are well within the abilitiesof those of ordinary skill in the art. The versatility of the labels setforth herein provides a marked advantage over currently utilized cyaninelabels, probes assembled using those labels and methods relying uponsuch labels and probes. Moreover, the present invention provides a classof chemically versatile labels in which the fluorophore can beengineered to have a desired light excitation and emission profile.

In a first aspect, the present invention provides a fluorescent compoundhaving the formula:

A and B independently selected monocyclic, bicyclic or polycyclic arylor heteroaryl moieties. When A and/or B is a bicyclic polycyclic moiety,two or more of the rings are optionally fused. Exemplary polycyclicmoieties include indole and benzoindole. Q is a substituted orunsubstituted methine moiety (e.g., —(CH═C(R))_(c)—CH═), in which c isan integer selected from 1, 2, 3, 4, or 5 and R is an “alkyl groupsubstituent” as defined herein. When two or more R groups are present,they are optionally joined to form a ring. Each R^(w), R^(x), R^(y) andR^(z) is independently selected from those substituents set forth in theDefinitions section herein as “alkyl group substituents” and “aryl groupsubstituents.” The indices w and z are independently selected from theintegers from 0 to 6. In an exemplary embodiment, at least one of R^(w),R^(x), R^(y) and R^(z) is C(O)NR^(o)(CH₂)_(h)G in which G is a memberselected from SO₃H and CO₂H, R^(o) is H or substituted or unsubstitutedalkyl or heteroalkyl and the index h is an integer from 1 to 20. Inexemplary embodiments, at least 1, 2, 3, 4, 5, or 6 of R^(x), R^(y),R^(w) and R^(z) are alkylsulfonic acid or heteroalkylsulfonic acid andat least one of these moieties is alkylcarboxylic acid orheteroalkylcarboxylic acid. In exemplary embodiments, at least one ofR^(w), R^(x), R^(y) and R^(z) includes a water-soluble polymer (e.g.,poly(ethylene glycol)) component.

In various embodiments, at least one of R^(w), R^(x), R^(y) and R^(z) isfunctionalized with an additional dye moiety bonded to the cyanine dyecore shown above. In an exemplary embodiment, the additional dye moietyis bonded to the dye core through a linker, a polyvalent scaffold, or alinker-polyvalent scaffold conjugate.

In a further aspect, the invention provides a method of monitoring anenzyme reaction. The method generally comprises providing a reactionmixture comprising the enzyme and at least a first reactant composition.An exemplary reactant composition comprises a compound having acomponent that reacts with the enzyme, a fluorescent label component,and an adaptor or linker-adaptor component joining the reactantcomponent to the label component. The reaction mixture is thenilluminated to excite the fluorescent label component, and a fluorescentsignal from the reaction mixture characteristic of the enzyme reactionis detected.

The invention also provides methods of monitoring nucleic acid synthesisreactions. The methods comprise contacting a polymerase/template/primercomplex with a fluorescently labeled nucleotide or nucleotide analoghaving a nucleotide or nucleotide analog component, a fluorescent labelcomponent, and an adaptor or linker-adaptor component joining dienucleotide or nucleotide analog component to the label component. Acharacteristic signal from the fluorescent dye is then detected that isindicative of incorporation of the nucleotide or nucleotide analog intoa primer extension reaction.

In various embodiments, the present invention provides methods of usingthe compounds described herein for performing nucleic acid analyses, andparticularly nucleic acid sequence analyses. In various embodiments, thecompounds of the invention are used in single molecule nucleic acidsequencing. Exemplary methods of the invention comprise using a templatenucleic acid complexed with a polymerase enzyme in a template dependentpolymerization reaction to produce a nascent nucleic acid strand,contacting the polymerase and template nucleic acid with a compound ofthe invention, and detecting whether or not the compound or asubstructure thereof (e.g., a monophosphate nucleic acid) wasincorporated into the nascent strand during the polymerization reaction,and identifying a base in the template strand based upon incorporationof the compound. Preferably, the foregoing process is carried out so asto permit observation of individual nucleotide incorporation reactions,through the use of, for example, an optical confinement, that allowsobservation of an individual polymerase enzyme, or through the use of aheterogeneous assay system, where fluorophores released fromincorporated analogs are detected.

The compounds and compositions of the invention are of use in singlemolecule or single molecule real time DNA sequencing assays. Ofparticular note in this context is the ability provided by the inventionto design fluorophores with selected absorbance and emission propertiesincluding wavelength and intensity. The compounds of the inventionprovide for very versatile assay design. For example, according to thepresent invention a series of fluorophores of use in an assay arereadily designed to have selected absorbance and emission wavelengthsand emission intensities, allowing multiple fluorophores to be utilizedand distinguished in an assay. In exemplary embodiments, use ofcompounds of the invention in a multifluorophore assay, e.g., singlemolecule DNA sequencing, enhances assay performance by at least about10%, at least about 20% or at least about 30% over a similar assay usingcurrently available fluorophores.

Other aspects, embodiments and objects of the present invention will beapparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1E displays representative sulfonic acid-derivatizedcyanine dyes of the invention.

FIG. 2A is a generic structure of exemplary dyes of the invention and ofsubstituents on these dyes. When incorporated into a conjugate of theinvention, the conjugated dyes can be conjugated to one or moreadditional species, e.g., a polyvalent scaffold (e.g., into a FRETpair), conjugated to a nucleic acid or to a linker. FIG. 2B is atabulation of exemplary dyes according to the generic structure of FIG.2A.

FIG. 3A is a generic structure of exemplary dyes of the invention and ofsubstituents on these dyes. When incorporated into a conjugate of theinvention, the conjugated dyes can be conjugated to one or moreadditional species, e.g., a polyvalent scaffold (e.g., into a FRETpair), conjugated to a nucleic acid or to a linker. FIG. 3B is atabulation of exemplary dyes according to the generic structure of FIG.3A.

FIG. 4A is a generic structure of exemplary dyes of the invention and ofsubstituents on these dyes. When incorporated into a conjugate of theinvention, the conjugated dyes can be conjugated to one or moreadditional species, e.g., a polyvalent scaffold (e.g., into a FRETpair), conjugated to a nucleic acid or to a linker. FIG. 4B is atabulation of exemplary dyes according to the generic structure of FIG.4A.

FIG. 5A is a generic structure of exemplary dyes of the invention and ofsubstituents on these dyes. When incorporated into a conjugate of theinvention, the conjugated dyes can be conjugated to one or moreadditional species, e.g., a polyvalent scaffold (e.g., into a FRETpair), conjugated to a nucleic acid or to a linker. FIG. 5B is atabulation of exemplary dyes according to the generic structure of FIG.5A.

FIG. 6A is a generic structure of exemplary dyes of the invention and ofsubstituents on these dyes. When incorporated into a conjugate of theinvention, the conjugated dyes can be conjugated to one or moreadditional species, e.g., a polyvalent scaffold (e.g., into a FRETpair), conjugated to a nucleic acid or to a linker. FIG. 6B is atabulation of exemplary dyes according to the generic structure of FIG.6A.

FIG. 7A is a generic structure of exemplary dyes of the invention and ofsubstituents on these dyes. When incorporated into a conjugate of theinvention, the conjugated dyes can be conjugated to one or moreadditional species, e.g., a polyvalent scaffold (e.g., into a FRETpair), conjugated to a nucleic acid or to a linker. FIG. 7B and FIG. 7Cis a tabulation of exemplary dyes according to the generic structure ofFIG. 7A.

FIG. 8A and FIG. 8B display structures of exemplary monovalent andpolyvalent dye nucleic acid (polyphosphate) conjugates of the invention.

FIG. 9 displays a structure of an exemplary polyvalent dye nucleic acid(polyphosphate) conjugates of the invention.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

“FET”, as used herein, refers to “Fluorescence Energy Transfer.”

“FRET”, as used herein, refers to “Fluorescence Resonance EnergyTransfer.” These terms are used herein to refer to both radiative andnon-radiative energy transfer processes. For example, processes in whicha photon is emitted and those involving long-range electron transfer areincluded within these terms. Throughout this specification, both ofthese phenomena are subsumed under the general term “donor-acceptorenergy transfer.”

Any of the dyes set forth herein can be a component of an FET or FRETpair as either the donor or acceptor. Conjugating a compound of theinvention and a donor or acceptor fluorophore through reactivefunctional groups on the conjugation partners and an appropriate linker,adaptor, carrier molecule or a combination thereof is well within theabilities of those of skill in the art.

The symbol “R”, as used herein, refers to moiety which is a memberselected from the moieties defined in the following section, e.g.,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, etc. as well as those groups set forth assubstituents of these moieties.

Definitions

Where chemical moieties are specified by their conventional chemicalformulae, written from left to right, they optionally equally encompassthe moiety which would result from writing the structure from right toleft, e.g., —CH₂O— is intended to also recite —OCH₂—; —NHS(O)₂— is alsointended to optionally represent. —S(O)₂HN—, etc. Moreover, wherecompounds can be represented as free acids or free bases or saltsthereof, the representation of a particular form, e.g., carboxylic orsulfonic acid, also discloses the other form, e.g., the deprotonatedsalt form, e.g., the carboxylate or sulfonate salt. Appropriatecounterions for salts are well-known in the art, and the choice of aparticular counterion for a salt of the invention is well within theabilities of those of skill in the art. Similarly, where the salt isdisclosed, this structure also discloses the compound in a free acid orfree base form. Methods of making salts and free acids and free basesare well-known in the art.

“Amino Acid,” as used herein refers to the genus encompassinghydrophilic amino acids, acidic amino acids, basic amino acids, polaramino acids, hydrophobic amino acids, aromatic amino acids, non-polaramino acids and aliphatic amino acids, including the genus and thespecies therein. The peptide linkers of the invention are formed fromsuch amino acids. Amino acids also encompass amino-carboxylic acidspecies other than α-amino acids, e.g., aminobutyric acid (aba),aminohexanoic acid(aha), aminomethylbenzoic acid (amb) etc.

“Hydrophilic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilic amino acids include Thr (T),Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) and Arg I.

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Glu (E) andAsp (D).

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Genetically encoded basic amino acids include His(H), Arg I and Lys (K).

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but which has at least onebond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (Q), Ser (S) and Thr (T).

“Hydrophobic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol.179:125-142. Exemplary hydrophobic amino acids include Ile (I), Phe (F),Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), Pro (P),and proline analogues.

“Aromatic Amino Acid” refers to a hydrophobic amino acid with a sidechain having at least one aromatic or heteroaromatic ring. The aromaticor heteroaromatic ring may contain one or more substituents such as —OH,—SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂, —NHR, —NRR, —C(O)R,—C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR and the like where each Ris independently (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl, (C₁-C₆)alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted(C₁-C₆) alkynyl, (C₁-C₂₁)) aryl, substituted (C₅-C₂₀) aryl, (C₆-C₂₆)alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl,substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl orsubstituted 6-26 membered alkheteroaryl. Genetically encoded aromaticamino acids include Phe (F), Tyr (Y) and Trp (W).

“Nonpolar Amino Acid” refers to a hydrophobic amino acid having a sidechain that is uncharged at physiological pH and which has bonds in whichthe pair of electrons shared in common by two atoms is generally heldequally by each of the two atoms (i.e., the side chain is not polar).Genetically encoded apolar amino acids include Leu (L), Val (V), Ile(I), Met (M), Gly (G) and Ala (A).

“Aliphatic Amino Acid” refers to a hydrophobic amino acid having analiphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala (A), Val (V), Leu (L) and Ile (I).

Peptide linkers in the compounds of the invention are formed from aminoacids linked by one or more peptide bond. The linkers are formed fromoligomers of the same amino acid or different amino acids.

An “Adaptor” is a moiety that is at least bivalent. Exemplary adaptorsare bound to a nucleic acid and a fluorescent dye, either directly orthrough a linker. The adaptor can also be bound to a second fluorescentdye, to a polyvalent scaffold or to a second nucleic acid. When theadaptor is bound to a second dye, either directly or through apolyvalent scaffold, the resulting conjugate is optionally a FRET pair.The adaptor is preferably bound to the phosphorus atom of a phosphate,phosphate ester or polyphosphate moiety of a nucleic acid. In exemplaryembodiments, the adaptor is bound through an amide moiety to the dye orto the linker of the linker-dye cassette. The amide moiety is formedbetween an amine on the adaptor and a carboxyl group on the dye or thelinker precursor.

“Cyanine,” as used herein, refers to aryl and heteroaryl polymethinedyes such as those based upon the cyanine, merocyanine, styryl andoxonol ring.

As used herein, “nucleic acid” means any natural or non-naturalnucleoside, or nucleotide and oligomers and polymers thereof, e.g., DNA,RNA, single-stranded, double-stranded, triple-stranded or more highlyaggregated hybridization motifs, and any chemical modifications thereof.Modifications include, but are not limited to, conjugation into acompound of the invention. Further modifications include those providingthe nucleic acid with a group that incorporates additional charge,polarizability, hydrogen bonding, electrostatic interaction,fluxionality or functionality to the nucleic acid. Exemplarymodifications include the attachment to the nucleic acid, at anyposition, of one or more hydrophobic or hydrophilic moieties, minorgroove binders, intercalating agents, quenchers, chelating agents, metalchelates, solid supports, and other groups that are usefully attached tonucleic acids. Exemplary nucleic acids of the invention include one ormore dye moiety of the invention bound thereto.

Exemplary modified nucleic acids include, but are not limited to,peptide nucleic acids (PNAs), those with phosphodiester groupmodifications (e.g., replacement of O⁻ with OR, NR, or SR), 2′-, 3′- and5′-position sugar modifications, modifications to the nucleobase moiety,e.g., 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, i.e., substitution of P(O)O₃ with another moiety,methylations, unusual base-pairing combinations such as the isobases,isocytidine and isoguanidine and the like. Nucleic acids can alsoinclude non-natural bases, e.g., nitroindole. Non-natural nucleobasesinclude bases that are modified with a compound of the invention or alinker-compound of the invention construct, a minor groove binder, anintercalating agent, a hybridization enhancer, a chelating agent, ametal chelate, a quencher, a fluorophore, a fluorogenic compound, etc.Modifications within the scope of “nucleic acid” also include 3′ and 5′modifications with one or more of the species described above.

The nucleic acid can comprise DNA, RNA or chimeric mixtures orderivatives or modified versions thereof. Both the probe and targetnucleic acid can be present as a single strand, duplex, triplex, etc.Moreover, as discussed above, the nucleic acid can be modified at thenucleobase moiety, sugar moiety, or phosphate backbone with other groupssuch as radioactive labels, minor groove binders, intercalating agents,donor and/or acceptor moieties and the like.

In addition to the naturally occurring “nucleobases,” adenine, cytosine,guanine and thymine, nucleic acid components of the compounds of theinvention optionally include modified bases. These components can alsoinclude modified sugars. For example, the nucleic acid can comprise atleast one modified base moiety which is selected from the groupincluding, but not limited to, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,nitroindole, and 2,6-diaminopurine. The dye of the invention or anotherprobe component can be attached to the modified base.

In another embodiment, the nucleic acid comprises at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose. The dye or anotherprobe component can be attached to the modified sugar moiety.

In yet another embodiment, the nucleic acid comprises at least onemodified phosphate backbone selected from the group including, but notlimited to, a peptide nucleic acid hybrid, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof. The dye or another probe component can beattached to the modified phosphate backbone.

“Nucleic acid” also includes a component of a conjugte with one or moremodified phosphate bridges (e.g., P(O)O₃) by conjugating a linker-dyeconjugate of the invention to the nucleic acid, e.g., replacing orderivatizing an oxygen of the bridge) with a compound of the inventionor a species that includes a compound of the invention attached to anadaptor. For example, “nucleic acid” also refers to species in which,rather than the P(O)(O⁻)O₂ moiety of a naturally occurring nucleic acid,includes the moiety ROP(O)(O—)O, in which R is a dye-linker conjugate ofthe invention, an adaptor, a linker-adaptor cassette or a fluorescentdye-linker-adaptor cassette. An exemplary linker is an amino acid orpeptide linker of the invention. In various embodiments, one oxygen ofthis structure is bound to the phosphorus atom of a P(O)(O⁻)O₂, suchthat the nucleic acid includes two or more phosphate moieties bound toeach other.

Further exemplary nucleic acids of the invention include a nucleotidehaving a polyphosphate moiety, e.g., pyrophosphate or a higherhomologue, such as the 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer and thelike. Exemplary nucleic acids include such a polyphosphate moiety bondedto the 5′-oxygen of a nucleoside. In addition to the attachedpolyphosphate moiety can include a modified phosphate bridge, such asthose exemplified herein. In an exemplary embodiment, the modifiedphosphate bridge is modified with an adaptor, a linker dye conjugate, alinker-adaptor cassette or a fluorescent dye-linker-adaptor cassette. Inan exemplary embodiment, the linker is an amino acid or peptide linkersuch as those set forth herein. Examples of some nucleic acids findinguse in the present invention are set forth in Published U.S. PatentApplication Nos. 2003/0124576 and 2007/0072196 as well as U.S. Pat. Nos.7,223,541 and 7,052,839, the full disclosures of which are incorporatedherein by reference for all purposes.

Furthermore, “nucleic acid” includes those species in which one or moreinternucleotide bridge does not include phosphorus: the bridge beingoptionally modified with a compound of the invention or a linker-dyeconstruct of the invention. An exemplary bridge includes a substitutedor unsubstituted alkyl or substituted or unsubstituted heteroalkylmoiety in which a carbon atom is the locus for the interconnection oftwo nucleoside sugar residues (or linker moieties attached thereto) anda linker-dye construct of the invention. The discussion above is notlimited to moieties that include a carbon atom as the point ofattachment; the locus can also be another appropriate linking atom, suchas nitrogen or another atom.

Phosphodiester linked nucleic acids of the invention can be synthesizedby standard methods known in the art, e.g. by use of an automated DNAsynthesizer using commercially available amidite chemistries (Ozaki etal., Nucleic Acids Research, 20: 5205-5214 (1992); Agrawal et al.,Nucleic Acids Research, 18: 5419-5423 (1990); Beaucage et al.,Tetrahedron, 48: 2223-2311 (1992); Molko et al., U.S. Pat. No.4,980,460; Koster et al., U.S. Pat. No. 4,725,677; Caruthers et al.,U.S. Pat. Nos. 4,415,732; 4,458,066; and 4,973,679). Nucleic acidsbearing modified phosphodiester linking groups can be synthesized bymethods known in the art. For example, phosphorothioate nucleic acidsmay be synthesized by the method of Stein et al. (Nucl. Acids Res.16:3209 (1988)), methylphosphonate nucleic acids can be prepared by useof controlled pore glass polymer supports (Sarin et al., Proc. Natl.Acad. Sci. U.S.A. 85:7448-7451 (1988)). Other methods of synthesizingboth phosphodiester- and modified phosphodiester-linked nucleic acidswill be apparent to those of skill in the art.

As used herein, “quenching group” refers to any fluorescence-modifyinggroup of the invention that can attenuate, at least partly, the energy(e.g., light) emitted by a fluorescent dye. This attenuation is referredto herein as “quenching”. Hence, irradiation of the fluorescent dye inthe presence of the quenching group leads to an emission signal from thefluorescent dye that is less intense than expected, or even completelyabsent. Quenching typically occurs through energy transfer between thefluorescent dye and the quenching group.

“Carrier molecule,” as used herein refers to any molecule to which acompound of the invention, or a conjugate incorporating a compound ofthe invention, is attached. Representative carrier molecules include anucleic acid, protein (e.g., enzyme, antibody), glycoprotein, peptide,saccharide (e.g., mono-, oligo-, and poly-saccharides), hormone,receptor, antigen, substrate, metabolite, transition state analog,cofactor, inhibitor, drug, dye, nutrient, growth factor, etc., withoutlimitation. “Carrier molecule” also refers to species that might not beconsidered to fall within the classical definition of “a molecule,”e.g., solid support (e.g., synthesis support, chromatographic support,membrane), virus and microorganism. An exemplary carrier molecule of usein the present invention is a polyphosphate nucleic acid. Exemplaryconjugates between a fluorescent dye and a polyphosphate nucleic acidare conjugated by covalent binding of the dye to the linker and hence tothe nucleic acid, or covalent binding of the dye to a linker and thelinker to the adaptor—the adaptor is conjugated to the nucleic acid.Alternatively, the dye is bound to a linker, which is bound to anadaptor, which is bound to the nucleic acid. In an exemplary embodiment,the adaptor is bound to the polyphosphate moiety through aphosphodiester bond. In an exemplary embodiment, the adaptor (or linker)is attached to the dye through a bond formed with an activatedderivative of a carboxyl moiety on the dye. In various embodiments, thebond is an amide bond.

“Activated derivatives of carboxyl moieties,” and equivalent species,refers to moiety on a precursor component of a conjugate of theinvention (e.g., dye, adaptor, linker, polyvalent moiety) having anoxygen-containing, or other, leaving group, e.g., an active ester, acylhalide, acyl imidazolide, etc.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturated alkylradicals include, but are not limited to, groups such as methyl,methylene, ethyl, ethylene, n-propyl, isopropyl, n-butyl, t-butyl,isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl,homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,n-octyl, and the like. An unsaturated alkyl group is one having one ormore double bonds or triple bonds. Examples of unsaturated alkyl groupsinclude, but are not limited to, vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. The term “alkyl,” unless otherwise noted, includes “alkylene”and, optionally, those derivatives of alkyl defined in more detailbelow, such as “heteroalkyl.”

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, S, P and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Also included aredi- and multi-valent species such as “cycloalkylene.” Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is meant to include, but not be limited to, speciessuch as trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings), which are fused togetheror linked covalently. The term “heteroaryl” refers to aryl groups (orrings) that contain from one to four heteroatoms selected from N, O, andS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. A heteroaryl group canbe attached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Also included are di- and multi-valentlinker species, such as “arylene.” Substituents for each of the abovenoted aryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) include both substituted and unsubstituted forms of theindicated radical. Exemplary substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, SO₃R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. Accordingly,from the above discussion of substituents, one of skill in the art willunderstand that the terms “substituted alkyl” and “heteroalkyl” aremeant to include groups that have carbon atoms bound to groups otherthan hydrogen atoms, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl(e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The substituents set forth in the paragraph above are referred to hereinas “alkyl group substituents.”

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″,—SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, SO₃R, —S(O)₂NR′R″, —NRSO₂R′, —CNand —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R″″are preferably independently selected from hydrogen, (C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl and heteroaryl, (unsubstitutedaryl)-(C₁-C₄)alkyl, and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

The substituents set forth in the two paragraphs above are referred toherein as “aryl group substituents.”

“Analyte”, “target”, “substance to be assayed”, and “target species,” asutilized herein refer to the species of interest in an assay mixture.The terms refer to a substance, which is detected qualitatively orquantitatively using a material, process or device of the presentinvention. Examples of such substances include cells and portionsthereof, enzymes, antibodies, antibody fragments and other biomolecules,e.g., antigens, polypeptides, glycoproteins, polysaccharides, complexglycolipids, nucleic acids, effector molecules, receptor molecules,enzymes, inhibitors and the like and drugs, pesticides, herbicides,agents of war and other bioactive agents.

More illustratively, such substances include, but are not limited to,tumor markers such as α-fetoprotein, carcinoembryonic antigen (CEA), CA125, CA 19-9 and the like; various proteins, glycoproteins and complexglycolipids such as (β₂-microglobulin (β₂ m), ferritin and the like;various hormones such as estradiol (E₂), estriol (E₃), human chorionicgonadotropin (hCG), luteinizing hormone (LH), human placental lactogen(hPL) and the like; various virus-related antigens and virus-relatedantibody molecules such as HBs antigen, anti-HBs antibody, HBc antigen,anti-HBc antibody, anti-HCV antibody, anti-HIV antibody and the like;various allergens and their corresponding IgE antibody molecules;narcotic drugs and medical drugs and metabolic products thereof; andnucleic acids having virus- and tumor-related polynucleotide sequences.

The term, “assay mixture,” refers to a mixture that includes the analyteand other components. The other components are, for example, diluents,buffers, detergents, and contaminating species, debris and the like thatare found mixed with the target. Illustrative examples include urine,sera, blood plasma, total blood, saliva, tear fluid, cerebrospinalfluid, secretory fluids from nipples and the like. Also included aresolid, gel or sol substances such as mucus, body tissues, cells and thelike suspended or dissolved in liquid materials such as buffers,extractants, solvents and the like.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like. Peptides can have mixed sequencesof be composed of a single amino acid, e.g., poly(lysine). An exemplarypolysaccharide is poly(sialic acid). An exemplary poly(ether) ispoly(ethylene glycol), e.g., m-PEG. Poly(ethylene imine) is an exemplarypolyamine, and poly(acrylic) acid is a representative poly(carboxylicacid).

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (i.e. PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(-PEG-OH).sub.m in which R represents the core moiety,such as glycerol or pentaerythritol, and m represents the number ofarms. Multi-armed PEG molecules, such as those described in U.S. Pat.No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as poly(propylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxypropylmethacrylamide), poly(α-hydroxy acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),such as described in U.S. Pat. No. 5,629,384, which is incorporated byreference herein in its entirety, and copolymers, terpolymers, andmixtures thereof. Although the molecular weight of each chain of thepolymer backbone can vary, it is typically in the range of from about100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000Da.

The term PEG or poly(ethylene glycol) is intended to be inclusive andnot exclusive. The term PEG includes poly(ethylene glycol) in any of itsforms, including alkoxy PEG, difunctional PEG, multiarmed PEG, forkedPEG, branched PEG, pendent PEG (i.e., PEG or related polymers having oneor more functional groups pendent to the polymer backbone), or PEG withdegradable linkages therein.

The PEG backbone can be linear or branched. Branched polymer backbonesare generally known in the art. Typically, a branched polymer has acentral branch core moiety and a plurality of linear polymer chainslinked to the central branch core. PEG is commonly used in branchedforms that can be prepared by addition of ethylene oxide to variouspolyols, such as glycerol, pentaerythritol and sorbitol. The centralbranch moiety can also be derived from several amino acids, such aslysine. The branched poly(ethylene glycol) can be represented in generalform as R(-PEG-OH)_(m) in which R represents the core moiety, such asglycerol or pentaerythritol, and m represents the number of arms.Multi-armed PEG molecules, such as those described in U.S. Pat. No.5,932,462, which is incorporated by reference herein in its entirety,can also be used as the polymer backbone.

An “Adaptor” is a moiety that is at least bivalent and which is bound toa linker bound to a dye or it is bound directly to the dye. The adaptoralso forms a bond with a second dye, polyvalent scaffold or to a nucleicacid. When the adaptor is bound to another dye, either directly orthrough a polyvalent scaffold, the resulting conjugate is optionally aFRET pair. When the adaptor is bound to a nucleic acid, it is preferablybound to the phosphorus atom of a phosphate, phosphate ester orpolyphosphate moiety. In exemplary embodiments, the adaptor is boundthrough an amide moiety to the dye. The amide moiety is formed betweenan amine on the adaptor and a carboxyl group on the dye.

“Readlength” is the number of bases the DNA polymerase enzyme at thebottom of the ZMW goes through during sequencing. A longer readlength isdesirable. Readlength depends, inter alia, on how fast the enzyme canincorporate fluorescent nucleotides of different colors (monitored thisby observing pulse widths and interpulse distances). Readlength alsodepends on how long the enzyme can incorporate analog without beingphotodamaged (damaged via undesired interactions with fluorescentnucleotides excited by light).

“Accuracy” is how precise a nucleotide with a base of a particular typecan be identified as the polymerase enzyme goes through incorporation offluorescent nucleotides. The base is identified by a pulse of a selectedwavelength upon incorporation of the nucleotide incorporating that base.Robust applications include precise base calling. Accuracy can bediminished by one or more of extra pulses, missing pulses and miscalledpulses.

“Extra pulses”—when a pulse is called and there is no nucleotideincorporation event. Extra pulses may be caused by branching (whenenzyme samples the fluorescent analog but does not incorporate), sticks(non-specific interactions of fluorescent nucleotides with enzymeoutside of incorporating site and surface of ZMW), photophysicalblinking (photophysically unstable behavior of fluorescent nucleotidesduring incorporation resulting in splitting of fluorescent signal).

“Missing pulses”—when a pulse is not called when there is in fact anucletided incorporation event. Missing pulses may be caused byinsufficient brightness of fluorescent nucleotides, low purity offluorescent nucleotides, or polymerase going too fast to detect allpulses.

“Miscalled pulses”—when pulse of different kind is called instead ofcorrect one. Miscalls may be caused by insufficient spectral separationbetween fluorescent nucleotides of different colors, photophysicalinstability of our fluorescent nucleotides, low intensity or highbackground of fluorescent nucleotide signal.

INTRODUCTION

The present invention provides a class of reactive fluorescent compoundsbased upon the cyanine-dye nucleus. Also provided is a wide variety ofconjugates of the cyanine dyes with, polyphosphate nucleotides, nucleicacids and other carrier molecules, including biological, non-biologicaland biologically active species. Selected cyanine labels describedherein include a functionalized linker arm that is readily convertedinto an array of reactive derivatives without requiring a modificationof the cyanine nucleus. Accordingly, the compounds of the inventionprovide an, as yet, undisclosed advantage, allowing facile access to anarray of conjugates between the linker arm-derivatized cyanine nucleusand carrier molecules.

Residing in the field of fluorescent labels, the present inventionprovides benefits of particular note. Fluorescent labels have theadvantage of requiring few precautions in handling, and being amenableto high-throughput visualization techniques (optical analysis includingdigitization of the image for analysis in an integrated systemcomprising a computer). Exemplary labels exhibit one or more of thefollowing characteristics: high sensitivity, high stability, lowbackground, low environmental sensitivity, high specificity in labeling,and a broader range of excitation/emission spectra. Many fluorescentlabels based upon the cyanine-nucleus are commercially available fromthe SIGMA chemical company (Saint Louis, Mo.), Molecular Probes (Eugene,Oreg.), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.),Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), GlenResearch, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.),Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs,Switzerland), and Applied Biosystems (Foster City, Calif.), as well asmany other commercial sources known to one of skill. Furthermore, thoseof skill in the art will recognize how to select an appropriatecyanine-based fluorophore for a particular application and, if it notreadily available commercially, will be able to synthesize the necessaryfluorophore de novo or synthetically modify commercially availablecyanine compounds to arrive at the desired fluorescent label.

The compounds, probes and methods discussed in the following sectionsare generally representative of the compositions of the invention andthe methods in which such compositions can be used. The followingdiscussion is intended as illustrative of selected aspects andembodiments of the present invention and it should not be interpreted aslimiting the scope of the present invention.

The Embodiments

Exemplary cyanine dyes in the compounds of the invention have theformula:

in which A and B are independently selected from substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl such thatthe compound is a fluorescent dye. When A and/or B is a bicyclicpolycyclic moiety, two or more of the rings are optionally fused.Exemplary polycyclic moieties include indole and benzoindole. Q is asubstituted or unsubstituted methine moiety (e.g., —(CH═C(R))_(c)—CH═),in which c is an integer selected from 1, 2, 3, 4, or 5 and each R isindependently H or an “alkyl group substituent” as defined herein). EachR^(w), R^(x), R^(y) and R^(z) is independently selected from thosesubstituents set forth in the Definitions section herein as “alkyl groupsubstituents” and “aryl group substituents” without limitation and inany combination. In various embodiments, one or more of R^(w), R^(x),R^(y) and R^(z) includes a poly(ethylene glycol) moiety. The indices wand z are independently selected from the integers from 0, 1, 2, 3, 4,5, 6 or greater. In an exemplary embodiment, at least one of R^(w),R^(x), R^(y) and R^(z) is —(CH₂)_(h)G in which G is a ionizable groupsuch as a member selected from SO₃H and CO₂H, and the index h is theinteger 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or greater. In exemplary embodiments, at least 1, 2, 3, 4, 5,or 6 of R^(x), R^(y), R^(w) and R^(z) are independently selectedalkylsulfonic acid or heteroalkylsulfonic acid and at least one of thesemoieties is alkylcarboxylic acid, heteroalkylcaboxylic acid,alkylsulfonic acid, and/or heteroalkylsulfonic acid.

In various embodiments, at least one of R^(w), R^(x), R^(y) and R^(z) isfunctionalized with an additional dye moiety bonded to the cyanine dyecore shown above. In an exemplary embodiment, the additional dye moietyis bonded to the dye core through a linker, a polyvalent scaffold, or alinker-polyvalent scaffold conjugate.

In various embodiments, the invention provides a compound having aformula selected from:

wherein Q is a methine moiety having a formula selected from:

in which one or more of the positions 1, 2, and 3 of the methine moietyis optionally substituted with Ar (e.g., R^(m)). The index n is selectedfrom the integers 1, 2, 3, 4, 5 and greater. The symbol Ar representssubstituted or unsubstituted aryl or substituted or unsubstitutedheteroaryl. Exemplary heteroaryl moieties are nitrogen containingheteroaryl moieties. An exemplary Ar moiety is a phenyl or pyridylmoiety substituted with one or more carboxylic acid, ester or amide. Theindices a and b are integers independently selected from 0, 1, 2, 3, and4.

The symbols R^(c), and R^(d) represent members independently selectedfrom alkyl and heteroalkyl, independently substituted with a memberselected from sulfonic acid, carboxylic acid, phosphonic acid, andphosphoric acid. The indices a, b, e and j are independently selectedfrom the integers 0, 1, 2, 3 and 4. Q is a methine linker and inexemplary embodiments is selected from:

in which n is the integer 1, 2 or 3.

In various embodiments, R^(a), R^(b), R^(e) and R^(j) represent moietiesthat are independently selected from H, halogen, C(O)R⁹, OR¹², NR¹²R¹³,CR¹²C(O)R¹³, NR¹²C(O)₂R¹³, SO₃H, and C(O)NR¹²R¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and substituted or unsubstituted heterocycloalkyl.

Exemplary R⁹ groups include OR¹⁰, and NH(CH₂)_(t)OR¹¹ in which R¹⁰ is amember selected from H and substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl. The index t is selected fromthe integers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or greater.

In various embodiments, R¹¹ is a member selected from H, and

in which the index u is selected from the integers 1, 2, 3, 4, 5, 6, 7,8 or greater. The symbol Y represents a nucleobase; and R¹² and R¹³ areindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl and substituted orunsubstituted heterocycloalkyl.

In various embodiments, R¹² and R¹³ are independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl and substituted or unsubstitutedheterocycloalkyl.

In various embodiments two or more of R^(a) moieties, R^(b) moieties,R^(e), moieties and/or R^(j), moieties together with the atoms to whichthey are attached are joined to form a ring structure which is selectedfrom a substituted or unsubstituted heterocylcoalkyl, substituted orunsubstituted aryl and a substituted or unsubstituted heteroaryl ringsystem. An exemplary substituted or unsubstituted aryl or heteroarylring is one substituted by at least one SO₃H, COOH, alkyl-SO₃H oralkyl-COOH moiety.

In various embodiments, R^(a), R^(b), R^(e) and R^(j) are independentlyselected from H, halogen, haloalkyl, SO₃H, alkyl sulfonic acid,alkylcarboxylic acid, sulfonamidoalkylsulfonic acid, amidoalkylsulfonicacid, amidoalkylcarboxylic acid, amidoalkylsulfonic acid,acyloxyalkylsulfonic acid, acyloxyalkylsulfonic acid, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl (e.g.,pyridyl), a moiety comprising poly(ethylene glycol), and a substitutedor unsubstituted aryl ring. The aryl ring is optionally formed byjoining two or more R^(a) moieties, two or more R^(b) moieties, two ormore R^(e) moieties or two or more R^(j) moieties. In variousembodiments, the ring structure is selected from substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl. Theinvention contemplates the full range of permutations of substituents atthe different ring positions as set forth above.

In various embodiments, at least two, at least three, or four of R², R³,R⁵ and R⁶ are alkylsulfonic acid. In exemplary embodiments, neither R⁴nor R⁸ is unsubstituted alkyl (e.g., not methyl). In variousembodiments, one of R⁴ and R⁸ is alkylcarboxylic acid and the other isalkylsulfonic acid. In various embodiments, when two or more R^(a)moieties are not joined to form an aryl ring substituted with at leastone SO₃H moiety, at least one R^(a) moiety is SO₃H. In exemplaryembodiments, when two or more R^(e) moieties are not joined to form anaryl ring substituted with at least one SO₃H moiety, at least one R^(e)is SO₃H. In various embodiments, none of R², R³, R⁵ and R⁶ isunsubstituted alkyl (e.g., none is C₁-C₄ unsubstituted alkyl, e.g., noneis methyl).

In exemplary embodiments, the invention provides compounds in which oneof R^(a), R^(b), R^(e) and R^(j) has the formula:

wherein R²⁰ and R²¹ are members independently selected from H, C(O)R¹⁴,OR¹⁵, NR¹⁵R¹⁶, CR¹⁵C(O)R¹⁶, NR¹⁵C(O)₂R¹⁶, SO₃H, and C(O)NR¹⁵R¹⁶,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl and substituted or unsubstitutedheterocycloalkyl.

The symbol R¹⁴ represents a member selected from H, OR³⁰, andsubstituted or unsubstituted alkyl. R¹⁵, R¹⁶, and R³⁰ are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl and substituted orunsubstituted heterocycloalkyl. R²² is a linker selected fromsubstituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl.

Exemplary compounds according to the invention are those wherein atleast one of R²⁰ and R²¹ is selected from C(O)NR²⁶R²⁷, and C(O)OR³⁰. Thesymbols R²⁶ and R²⁷ are independently members selected fromalkylsulfonic acid, heteroalkylsulfonic acid, alkylcarboxylic acid,heteroalkylcarboxylic acid; however, when one is alkylsulfonic acid orheteroalkylsulfonic acid, the other is optionally H. R³⁰ is selectedfrom alkylsulfonic and heteroalkylsulfonic acid.

In various embodiments, at least one of R^(a), R^(b), R^(e) and R^(j)has the formula:

in which each Z is independently selected from SO₃H and CO₂H, each x isan integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or greater and each R^(N) is independently selected from H, substitutedor unsubstituted alkyl, e.g., C₁-C₁ substituted or unsubstituted alkyl.

In various embodiments, at least one of R^(a), R^(b), R^(e) and R^(j) isindependently selected from: H, OCH₃, SO₃H, COOH,

TABLE A

in which the index a is 0 or 1; the index x represents the integer 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. When a structure includes morethan one x, the value of each x is independently selected. The index mis an integer which provides a polyethylene glycol moiety of molecularweight at least about 100, at least about 300, at least about 500, atleast about 1000, or at least about 5000 daltons. Rp is H, substitutedor unsubstituted alkylr or substituted or unsubstituted heteroalkyl. R8is a member selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl. The position of substitutionon the pyridyl ring can vary and the point of attachment to the cyaninenucleus can be ortho, meta or para to the endocyclic nitrogen atom.

In an exemplary embodiment, at least one, preferably two or more ofR^(a), R^(b), R^(e) or R^(j) is:

As will be appreciated by those of skill in the art the alkyl alkylmoieties attached to COOH and SO₃H can be either shorter or longer thanshown (e.g., independently 1, 2, 3, 4, 5, 6, 7, 8 9, 10, 11 or 12), orcan be replaced with a water-soluble polymer moiety, e.g., poly(ethyleneglycol). This moiety can be used as a locus for attachment of anotherdye or a cassette including another dye, such as a linker-dye cassette.In an exemplary embodiment, the carboxylic acid is converted to an amidecomponent of the multidye construct:

In an exemplary embodiment, the amide is a component of a polyvalentscaffold to which the other dye is or dyes are attached. For example:

in which L^(f) is a linkage fragment, for example, S, SC(O)NH, HNC(O)S,SC(O)O, O, NH, NHC(O), (O)CNH and NHC(O)O, and OC(O)NH, CH₂S, CH₂O,CH₂CH₂O, CH₂CH₂S, (CH₂)_(o)O, (CH₂)_(o)S or (CH₂)_(o)Y′-PEG wherein, Y′is S, NH, NHC(O), C(O)NH, NHC(O)O, OC(O)NH, or O and o is an integerfrom 1 to 50. An exemplary “Dye” moiety is a cyanine dye, e.g., thosedisclosed or referenced herein. Similarly, a dye to which the pyridylsubstituent attached is a cyanine dye disclosed or referenced herein.

Exemplary compounds of the invention include:

As will be appreciated by those of skill in the art, the precedingdiscussion regarding the substituents, R^(a), R^(b), R^(e), R^(d),R^(e), R^(f), R^(g), R^(h), R^(i), R^(j) is fully applicable withrespect to the formulae above. For example R⁶, R^(6′), R⁷ and R^(7′)correspond to R^(f), R^(g), R^(h) and R^(i), respectively. Similarly, R⁵and R^(5′) correspond to R^(c) and R^(d). Depending on the ring to whichthey are joined, R¹-R⁴ and R^(1′)-R^(4′) correspond to R^(a), R^(e) andR^(j).

In various embodiments, R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴ andR^(4′) are independently selected from H, SO₃H, alkyl sulfonic acid,alkylcarboxylic acid, sulfonamidoalkylsulfonic acid, amidoalkylsulfonicacid, amidoalkylcarboxylic acid, amidoalkylsulfonic acid,acyloxyalkylsulfonic acid, acyloxyalkylsulfonic acid, and substituted orunsubstituted heteroaryl (e.g., pyridyl). Those substituents thatinclude an alkyl subunit, in exemplary embodiments, include 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or more carbon atoms in the alkyl chain of thealkyl subunit.

In various embodiments, at least two, at least three, or four of R¹,R^(1′), R², R^(2′), R³, R^(3′), R⁴ and R^(4′) are alkylsulfonic acid. Inexemplary embodiments, neither R⁵ nor R^(5′) is unsubstituted alkyl(e.g., not methyl). In various embodiments, one of R⁵ and R^(5′) isalkylcarboxylic acid and the other is alkylsulfonic acid. In variousembodiments, at least one of R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴ andR^(4′) is SO₃H. In various embodiments, none of R⁶, R^(6′), R⁷ andR^(7′) is unsubstituted alkyl (e.g., none is C₁-C₄ unsubstituted alkyl,e.g., none is methyl).

In various embodiments, the invention provides compounds in which amember selected from R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴ and R^(4′)is:

wherein the index z is selected from the integers 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or greater, and Z is a memberselected from H, C₁-C₄ alkyl (e.g., methyl), and —(CH₂)_(y)X, in which Xis CO₂H or SO₃H and the index y is selected from the integers 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or greater. Inexemplary embodiments, two or more of R¹, R^(1′), R², R^(2′), R³,R^(3′), R⁴ and R^(4′) is a moiety of this formula.

In various embodiments, at least two of R⁶, R^(6′), R⁷ and R^(7′) arealkylsulfonic acid. In exemplary embodiments, at least one of R⁶ andR^(6′) is a group other than unsubstituted alkyl (e.g., not methyl). Invarious embodiments, none of R⁶, R^(6′), R⁷ and R^(7′) is unsubstitutedalkyl (e.g., not methyl).

In various embodiments of the invention, the compounds have a formula inwhich 1, 2, 3, or 4 of R⁶, R^(6′), R⁷ and R^(7′) is:

wherein each f is an integer independently selected from 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or greater.

In exemplary embodiments, both R⁵ and R^(5′) are alkylsulfonic acid. Inexemplary embodiments, one of R⁵ and R^(5′) is alkylsulfonic acid andthe other is alkylcarboxylic acid. In various embodiments, the alkylmoiety is C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀ or longer.

These and additional cyanine dyes of use in practicing the instantinvention are set forth in commonly owned U.S. Provisional PatentApplication Nos. 61/377,048, titled “Cyanine Dyes”, 61/377,031, titled“Phospholinked Dye Analogs with an Amino Acid Linker”, 61/377,022,titled “Scaffold-Based Dyes”, and 61/377,004, titled “Molecular Adaptorsfor Dye Conjugates”. The disclosure of each of these applications isincorporated herein by reference in its entirety for all purposes.

In exemplary embodiments, the compounds of the invention include:

in which R⁴ and R^(4′) are independently selected from substituents setforth in Table A, supra. The indices b, x and m independendentlyrepresent the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17 and 18.

In various embodiments, the invention provides the compounds:

in which the index c is 0 or 1, the index n is the integer 0, 1, 2, 3,4, 5, or 6. R⁴ and R^(4′) are independently selected from substituentsset forth in Table A, supra.

In exemplary embodiments, the compounds of the invention include the abond between the dye and a carrier molecule (e.g., a nucleic acid,linker-nucleic acid or linker-adaptor nucleic acid). Exemplary compoundsaccording to this format include a C(O)— moiety derived from acarboxylic acid, such as:

As will be apparent to those of skill in the art, any of the carboxylicacid containing dyes of the invention can be conjugated to a carriermolecule in this manner.

In exemplary embodiments, the compounds of the invention have theformula:

As will be appreciated by those of skill in the art, the number ofphosphate moieties in these compounds can vary and can include 1, 2, 3,4, 5, 6, 7, 8, or more phosphates. Moreover, the number of CH₂ moietiesin the alkylamide adaptor between the dye and the polyphosphate can bevaried from 4 to 16, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or16.

Synthesis

An exemplary synthetic route to sulfonated cyanine dyes of the inventionis set forth in Scheme 1. Starting keto-ethyl ester 1 is converted tosulfonate 2, which is decarboxylated to sulfonic acid 3. The sulfonicacid is annulated with hydrazine 5 to form the sulfonic acid substitutedindolenine 6. At this stage, the indolenine nitrogen can be alkylatedwith, for example, an activated alkyl carboxylic acid or an activatedsulfonic acid to form compound 8 and 9, respectively. The methine groupis placed on the indolenine ring by the action of(E)-N-((E)-3-(phenylamino)allylidene)aniline, 10, to form 11. Compound11 is condensed with compound 8 in acetic anhydride/pyridine to providecyanine 12.

Chemical synthesis of nucleic acid derivatives of the cyanine dyes ofthe invention is generally automated and is performed by couplingnucleosides through phosphorus-containing covalent linkages. The mostcommonly used oligonucleotide synthesis method involves reacting anucleoside with a protected cyanoethyl phosphoramidite monomer in thepresence of a weak acid. The coupling step is followed by oxidation ofthe resulting phosphite linkage. Finally, the cyanoethyl protectinggroup is removed and the nucleic acid is cleaved from the solid supporton which it was synthesized. The labels of the present invention can beincorporated during oligonucleotide synthesis using a mono- orbis-phosphoramidite derivative of the fluorescent compound of theinvention. Alternatively, the label can be introduced by combining acompound of the invention that includes a reactive functional group withthe nucleic acid under appropriate conditions to couple the compound tothe nucleic acid. In yet another embodiment, the fluorescent compound isattached to a solid support through a linker arm, such as a substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl or anucleic acid residue. Synthesis proceeds with the fluorescent moietyalready in place on the growing nucleic acid chain.

Enzymatic methods of synthesis involve the use of fluorescent-labelednucleic acids in conjunction with a nucleic acid template, a primer andan enzyme. Efficient enzymatic incorporation of a fluorescent-labelednucleic acid is facilitated by selection of reaction partners that donot adversely affect the enzymes ability to couple the partners.

In those embodiments of the invention in which the cyanine-basedfluorescent compound of the invention is attached to a nucleic acid, thecarrier molecule is produced by either synthetic (solid phase, liquidphase or a combination) or enzymatically or by a combination of theseprocesses.

Another synthetic strategy for the preparation of oligonucleotides isthe H-phosphonate method (B. Froehler and M. Matteucci, TetrahedronLett., vol 27, p 469-472, 1986). This method utilizes activatednucleoside H-phosphonate monomers rather than phosphoramidites to createthe phosphate internucleotide linkage. In contrast to thephosphoramidite method, the resulting phosphonate linkage does notrequire oxidation every cycle but instead only a single oxidation stepat the end of chain assembly. The H-phosphonate method may also be usedto conjugate reporters and dyes to synthetic oligonucleotide chains (N.Sinha and R. Cook, Nucleic Acids Research, Vol 16, p. 2659, 1988).

In an exemplary embodiment, the synthesis and purification of thenucleic acid conjugates of compounds of the invention results in ahighly pure conjugate, which, if it is a mixture, less than about 30% ofthe nucleic acid is unlabeled with a dye of the invention, preferablyless than about 20% are unlabeled, more preferably less than about 10%,still more preferably less than about 5%, more preferably less thanabout 1%, more preferably less than about 0.5%, or more preferably lessthan about 0.1% and even more preferably less than 0.01% of the nucleicacid is unlabeled with a cyanine dye of the invention. In certainembodiments, the nucleic acid (e.g., nucleotides and/or nucleotideanalogs) is incorporatable by a polymerase enzyme in atemplate-dependent polymerization reaction.

The compounds of the invention can be prepared as a single isomer or amixture of isomers, including, for example cis-isomers, trans-isomers,diastereomers and stereoisomers. In a preferred embodiment, thecompounds are prepared as substantially a single isomer. Isomericallypure compounds are prepared by using synthetic intermediates that areisomerically pure in combination with reactions that either leave thestereochemistry at a chiral center unchanged or result in its completeinversion. Alternatively, the final product or intermediates along thesynthetic route can be resolved into a single isomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose anappropriate resolution or synthetic method for a particular situation.See, generally, Furniss et al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICALORGANIC CHEMISTRY 5^(TH) ED., Longman Scientific and Technical Ltd.,Essex, 1991, pp. 809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

Reactive Functional Groups

The compounds of the invention are assembled from covalent bondingreactions between precursors bearing a reactive functional group, whichis a locus for formation of a covalent bond between the precursors. Theprecursors of compounds of the invention bear a reactive functionalgroup, which can be located at any position on the compound. Thefinished dye conjugates can include a further reactive functional groupat any point on the molecule.

Exemplary species include a reactive functional group attached directlyto a cyanine nucleus (e.g., aryl ring or methine bridge) or to a linkerattached to a component (e.g., aryl ring or methine bridge) of the dyemoiety. Other molecules include a reactive functional group attached toa polyvalent moiety. An exemplary reactive functional group is attachedto an alkyl or heteroalkyl moiety on the dye. When the reactive group isattached a substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl linker moiety, the reactive group ispreferably located at a terminal position of the alkyl or heteroalkylchain. Reactive groups and classes of reactions useful in practicing thepresent invention are generally those that are well known in the art ofbioconjugate chemistry. Currently favored classes of reactions availablewith reactive dye-based compounds of the invention are those proceedingunder relatively mild conditions. These include, but are not limited tonucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions) and additions to carbon-carbon and carbon-heteroatom multiplebonds (e.g., Michael reaction, Diels-Alder addition). These and otheruseful reactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982.

Useful Reactive Functional Groups Include, for Example:

-   -   (a) carboxyl groups and derivatives thereof including, but not        limited to activated esters, e.g., N-hydroxysuccinimide esters,        N-hydroxyphthalimide, N-hydroxybenztriazole esters, acid        halides, acyl imidazoles, thioesters, p-nitrophenyl esters,        alkyl, alkenyl, alkynyl and aromatic esters, activating groups        used in peptide synthesis and acid halides;    -   (b) hydroxyl groups, which can be converted to esters,        sulfonates, phosphoramidates, ethers, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be displaced with a        nucleophilic group such as, for example, an amine, a carboxylate        anion, thiol anion, carbanion, or an alkoxide ion, thereby        resulting in the covalent attachment of a new group at the site        of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, allowing derivatization via        formation of carbonyl derivatives, e.g., imines, hydrazones,        semicarbazones or oximes, or via such mechanisms as Grignard        addition or alkyllithium addition;    -   (f) sulfonyl halide groups for reaction with amines, for        example, to form sulfonamides;    -   (g) thiol groups, which can be converted to disulfides or        reacted with acyl halides, for example;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc;    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds; and    -   (k) phosphoramidites and other standard functional groups useful        in nucleic acid synthesis.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assembleor utilize the reactive dye analogue. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

In addition to those embodiments in which a compound of the invention isattached directly to a carrier molecule, the fluorophores can also beattached by indirect means. In various embodiments, a ligand molecule(e.g., biotin) is covalently bound to the probe species. The ligand thenbinds to another molecules (e.g., streptavidin) molecule, which iseither inherently detectable or covalently bound to a signal system,such as a fluorescent compound, or an enzyme that produces a fluorescentcompound by conversion of a non-fluorescent compound. Useful enzymes ofinterest as labels include, for example, hydrolases, particularlyphosphatases, esterases and glycosidases, hydrolases, peptidases oroxidases, and peroxidases.

Polyphosphate Analogues

In an exemplary embodiment, the present invention is generally directedto compositions that comprise compounds analogous to nucleotides, andwhich, in various aspects are readily processible by nucleic acidprocessing enzymes, such as polymerases. In addition to the unexpectedlyadvantageous features imparted to the compounds by incorporation of dyesof novel structure, the compounds of the invention generally benefitfrom one or more advantages of greater stability to undesired enzymaticor other cleavage or non-specific degradation, as well as incorporationefficiencies that are better than or at least comparable totriphosphate, tetraphosphate or pentaphosphate analogs. Exemplarypolyphosphates and their uses are set forth in commonly owned U.S. Pat.No. 7,405,281.

In various embodiments, the invention provides polyphosphate analogs ofthe cyanine dyes of the invention. In various embodiments, thepolyphosphate analogs are polyphosphate analogue of a nucleic acid. Anexemplary compound according to this motif has the general structure:

in which NA is the nucleic acid. The index u is an integer selected from1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In an exemplary embodiment, the polyphosphate analogue of the inventionhas the general structure:

in which Y is a naturally occurring or non-natural nucleobase.

In various embodiments, the polyphosphate analogue of the invention hasthe general structure:

in which t is an integer selected from 1-40, more particularly, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or higher.

In an exemplary embodiment, the polyphosphate analogue of the inventionhas the general structure:

As will be apparent to those of skill in the art, the component labeled“cyanine” refers to the cyanine dyes of the invention set forth hereinas well as known cyanines not known to have been conjugated into thestructures set forth herein.

In an exemplary embodiment, the cyanine dye component comprises multiplecyanine dyes bound to a common polyvalent scaffold or amplifier.Examples of such scaffold-based cyanine dyes are described in commonlyowned U.S. Provisional Patent Application No. 61/377,022, the disclosureof which is incorporated in its entirety herein by reference for allpurposes. Examples of dyes that can be incorporated with the dyes of theinstant invention into a scaffold-based dye are set forth in U.S.Provisional Patent Application No. 61/377,048, the disclosure of whichis incorporated in its entirety herein by reference for all purposes.The scaffold-based dyes of the invention can include FET or FRET pairs.In an exemplary embodiment, the scaffold-based dye composition includesa Cy3 and a Cy5 dye attached to a common polyvalent scaffold oramplifier. In various embodiments, the linker component includes apeptide component. Exemplary peptide components are set forth incommonly owned U.S. Provisional Patent Application No. 61/377,031, thedisclosure of which is incorporated in its entirety herein by referencefor all purposes. In various embodiments, the linker or cyanine dyecomponent includes an adaptor moiety as set forth in commonly owned U.S.Provisional Patent Application No. 61/377,004, the disclosure of whichis incorporated in its entirety herein by reference for all purposes.

Probes

The invention provides probes having a dye of the invention conjugatedto a carrier molecule, for example, a target species (e.g., receptor,enzyme, etc.) a ligand for a target species (e.g., nucleic acid,peptide, etc.), a small molecule (e.g., drug, pesticide, etc.), a solidsupport and the like. The probes can be used for in vitro and in vivoapplications. Exemplary probes are those in which the dye is conjugatedto the carrier molecule through an adaptor or through a linker-adaptorcassette.

Small Molecule Probes

The dyes of the invention can be used as components of small moleculeprobes. In an exemplary design, a small molecule probe includes a dye ofthe invention and a second species that alters the luminescentproperties of the dyes, e.g., a quencher of fluorescence. In anexemplary embodiment, an agent, such as an enzyme cleaves the dye of theinvention, the quencher or both from the small molecule generatingfluorescence in the system under investigation (see, for example,Zlokarnik et al., Science 279: 84-88 (1998)).

Nucleic Acid Capture Probes

In one embodiment, an immobilized nucleic acid comprising a dye of theinvention is used as a capture probe. The nucleic acid probe can be usedin solution phase or it can be attached to a solid support. Theimmobilized probes can be attached directly to the solid support orthrough a linker arm between the support and the dye or between thesupport and a nucleic acid residue. Preferably, the probe is attached tothe solid support by a linker (i.e., spacer arm, supra). The linkerserves to distance the probe from the solid support. The linker is mostpreferably from about 5 to about 30 atoms in length, more preferablyfrom about 10 to about 50 atoms in length. Exemplary attachment pointsinclude the 3′- or 5′-terminal nucleotide of the probe as well as otheraccessible sites discussed herein.

Chemical synthesis of nucleic acid probes containing a dye of theinvention is optionally automated and is performed by couplingnucleosides through phosphorus-containing covalent linkages. The mostcommonly used oligonucleotide synthesis method involves reacting anucleoside with a protected cyanoethyl phosphoramidite monomer in thepresence of a weak acid. The coupling step is followed by oxidation ofthe resulting phosphite linkage. Finally, the cyanoethyl protectinggroup is removed and the nucleic acid is cleaved from the solid supporton which it was synthesized. The labels of the present invention can beincorporated during oligonucleotide synthesis using a mono- orbis-phosphoramidite derivative of the fluorescent compound of theinvention. Alternatively, the label can be introduced by combining acompound of the invention that includes a reactive functional group withthe nucleic acid under appropriate conditions to couple the compound tothe nucleic acid. In yet another embodiment, the fluorescent compound isattached to a solid support through a linker arm, such as a substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl or anucleic acid residue. Synthesis proceeds with the fluorescent moietyalready in place on the growing nucleic acid chain.

Enzymatic methods of synthesis involve the use of fluorescent-labelednucleic acids in conjunction with a nucleic acid template, a primer andan enzyme. Efficient enzymatic incorporation of a fluorescent-labelednucleic acid is facilitated by selection of reaction partners that donot adversely affect the enzymes ability to couple the partners.

In those embodiments of the invention in which the dye-based fluorescentcompound of the invention is attached to a nucleic acid, the carriermolecule is produced by either synthetic (solid phase, liquid phase or acombination) or enzymatically or by a combination of these processes.

Another synthetic strategy for the preparation of oligonucleotides isthe H-phosphonate method (B. Froehler and M. Matteucci, TetrahedronLett., vol 27, p 469-472, 1986). This method utilizes activatednucleoside H-phosphonate monomers rather than phosphoramidites to createthe phosphate internucleotide linkage. In contrast to thephosphoramidite method, the resulting phosphonate linkage does notrequire oxidation every cycle but instead only a single oxidation stepat the end of chain assembly. The H-phosphonate method may also be usedto conjugate reporters and dyes to synthetic oligonucleotide chains (N.Sinha and R. Cook, Nucleic Acids Research, Vol 16, p. 2659, 1988).

In an exemplary embodiment, the synthesis and purification of thenucleic acid conjugates of compounds of the invention results in ahighly pure conjugate, which, if it is a mixture, less than about 30% ofthe nucleic acid is unlabeled with a dye of the invention, preferablyless than about 20% are unlabeled, more preferably less than about 10%,still more preferably less than about 5%, more preferably less thanabout 1%, more preferably less than about 0.5%, or more preferably lessthan about 0.1% and even more preferably less than 0.01% of the nucleicacid is unlabeled with a dye of the invention. In certain embodiments,the nucleic acid (e.g., nucleotides and/or nucleotide analogs) isincorporatable by a polymerase enzyme in a template-dependentpolymerization reaction.

Dual Labeled Probes

The present invention also provides dual labeled probes that includeboth a dye of the invention and another label. Exemplary dual labeledprobes include nucleic acid probes that include a nucleic acid with adye of the invention attached thereto, typically, through an adaptor oradaptor-linker cassette. Exemplary probes include both a dye of theinvention and a quencher. The probes are of use in a variety of assayformats. For example, when a nucleic acid singly labeled with a dye ofthe invention is the probe, the interaction between the first and secondnucleic acids can be detected by observing the interaction between thedye of the invention and the nucleic acid. Alternatively, theinteraction is the quenching by a quencher attached to the secondnucleic acid of the fluorescence from a dye of the invention.

The dyes of the invention are useful in conjunction with nucleic-acidprobes in a variety of nucleic acid amplification/quantificationstrategies including, for example, 5′-nuclease assay, StrandDisplacement Amplification (SDA), Nucleic Acid Sequence-BasedAmplification (NASBA), Rolling Circle Amplification (RCA), as well asfor direct detection of targets in solution phase or solid phase (e.g.,array) assays. Furthermore, the dye of the invention-derivatized nucleicacids can be used in probes of substantially any format, including, forexample, format selected from molecular beacons, Scorpion Probes™,Sunrise Probes™, conformationally assisted probes, light up probes,Invader Detection probes, and TaqMan™ probes. See, for example,Cardullo, R., et al., Proc. Natl. Acad. Sci. USA, 85:8790-8794 (1988);Dexter, D. L., J. Chem. Physics, 21:836-850 (1953); Hochstrasser, R. A.,et al., Biophysical Chemistry, 45:133-141 (1992); Selvin, P., Methods inEnzymology, 246:300-334 (1995); Steinberg, I., Ann. Rev. Biochem.,40:83-114 (1971); Stryer, L., Ann. Rev. Biochem., 47:819-846 (1978);Wang, G., et al., Tetrahedron Letters, 31:6493-6496 (1990); Wang, Y., etal., Anal. Chem., 67:1197-1203 (1995); Debouck, C., et al., insupplement to nature genetics, 21:48-50 (1999); Rehman, F. N., et al.,Nucleic Acids Research, 27:649-655 (1999); Cooper, J. P., et al.,Biochemistry, 29:9261-9268 (1990); Gibson, E. M., et al., GenomeMethods, 6:995-1001 (1996); Hochstrasser, R. A., et al., BiophysicalChemistry, 45:133-141 (1992); Holland, P. M., et al., Proc Natl. Acad.Sci USA, 88:7276-7289 (1991); Lee, L. G., et al., Nucleic Acids Rsch.,21:3761-3766 (1993); Livak, K. J., et al., PCR Methods and Applications,Cold Spring Harbor Press (1995); Vamosi, G., et al., BiophysicalJournal, 71:972-994 (1996); Wittwer, C. T., et al., Biotechniques,22:176-181 (1997); Wittwer, C. T., et al., Biotechniques, 22:130-38(1997); Giesendorf, B. A. J., et al., Clinical Chemistry, 44:482-486(1998); Kostrikis, L. G., et al., Science, 279:1228-1229 (1998); Matsuo,T., Biochemica et Biophysica Acta, 1379:178-184 (1998); Piatek, A. S.,et al., Nature Biotechnology, 16:359-363 (1998); Schofield, P., et al.,Appl. Environ. Microbiology, 63:1143-1147 (1997); Tyagi S., et al.,Nature Biotechnology, 16:49-53 (1998); Tyagi, S., et al., NatureBiotechnology, 14:303-308 (1996); Nazarenko, I. A., et al., NucleicAcids Research, 25:2516-2521 (1997); Uehara, H., et al., Biotechniques,26:552-558 (1999); D. Whitcombe, et al., Nature Biotechnology,17:804-807 (1999); Lyamichev, V., et al., Nature Biotechnology, 17:292(1999); Daubendiek, et al., Nature Biotechnology, 15:273-277 (1997);Lizardi, P. M., et al., Nature Genetics, 19:225-232 (1998); Walker, G.,et al., Nucleic Acids Res., 20:1691-1696 (1992); Walker, G. T., et al.,Clinical Chemistry, 42:9-13 (1996); and Compton, J., Nature, 350:91-92(1991).

In view of the well-developed body of literature concerning theconjugation of small molecules to nucleic acids, many other methods ofattaching donor/acceptor pairs to nucleic acids will be apparent tothose of skill in the art.

More specifically, there are many linking moieties and methodologies forattaching groups to the 5′- or 3′-termini of nucleic acids, asexemplified by the following references: Eckstein, editor, Nucleic acidsand Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckermanet al., Nucleic Acids Research, 15: 5305-5321 (1987) (3′-thiol group onnucleic acid); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′-sulfhydryl); Giusti et al., PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′-phosphoamino groupvia Aminolink TM II available from P.E. Biosystems, CA.) Stabinsky, U.S.Pat. No. 4,739,044 (3-aminoalkylphosphoryl group); Agrawal et al.,Tetrahedron Letters, 31: 1543-1546 (1990) (attachment viaphosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15:4837 (1987) (5-mercapto group); Nelson et al., Nucleic Acids Research,17: 7187-7194 (1989) (3′-amino group), and the like.

Exemplary fluorophores that can be combined in a probe or scaffold-baseddye with a dye of the invention include those set forth in Table 1.

TABLE 1 Exemplary Donors or Acceptors for Compounds of the Invention4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid acridine andderivatives:    acridine    acridine isothiocyanate5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonateN-(4-anilino-1-naphthyl)maleimide anthranilamide BODIPY Brilliant Yellowcoumarin and derivatives: coumarin    7-amino-4-methylcoumarin (AMC,Coumarin 120)    7-amino-4-trifluoromethylcouluarin (Coumaran 151)cyanine dyes cyanosine 4′,6-diaminidino-2-phenylindole (DAPI)5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red)7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride)4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC) eosin andderivatives:    eosin    eosin isothiocyanate erythrosin andderivatives:    erythrosin B    erythrosin isothiocyanate ethidiumfluorescein and derivatives:    5-carboxyfluorescein (FAM)   5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)   2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE)   fluorescein    fluorescein isothiocyanate    QFITC (XRITC)fluorescamine IR144 IR1446 Malachite Green isothiocyanate4-methylumbelliferone ortho cresolphthalein nitrotyrosine pararosanilinePhenol Red B-phycoerythrin o-phthaldialdehyde pyrene and derivatives:   pyrene butyrate    succinimidyl 1-pyrene butyrate quantum dotsReactive Red 4 (Cibacron ™ Brilliant Red 3B-A) rhodamine andderivatives:    6-carboxy-X-rhodamine (ROX)    6-carboxyrhodamine (R6G)   lissamine rhodamine B sulfonyl chloride rhodamine (Rhod)    rhodamineB    rhodamine 123    rhodamine X isothiocyanate    sulforhodamine B   sulforhodamine 101 sulfonyl chloride derivative of sulforhodamine 101(Texas Red) N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA) tetramethylrhodamine    tetramethyl rhodamine isothiocyanate (TRITC) riboflavinrosolic acid terbium chelate derivatives Black Hole Quenchers ™

There is a great deal of practical guidance available in the literaturefor functionalizing fluorophores and selecting appropriatedonor-acceptor pairs for particular probes, as exemplified by thefollowing references: Pesce et al., Eds., FLUORESCENCE SPECTROSCOPY(Marcel Dekker, New York, 1971); White et al., FLUORESCENCE ANALYSIS: APRACTICAL APPROACH (Marcel Dekker, New York, 1970); and the like. Theliterature also includes references providing exhaustive lists offluorescent and chromogenic molecules and their relevant opticalproperties for choosing reporter-quencher pairs (see, for example,Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES, 2ndEdition (Academic Press, New York, 1971); Griffiths, COLOUR ANDCONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York, 1976);Bishop, Ed., INDICATORS (Pergamon Press, Oxford, 1972); Haugland,HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes,Eugene, 1992) Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (IntersciencePublishers, New York, 1949); and the like. Further, there is extensiveguidance in the literature for derivatizing reporter and quenchermolecules for covalent attachment via common reactive groups that can beadded to a nucleic acid, as exemplified by the following references:Haugland (supra); Ullman et al., U.S. Pat. No. 3,996,345; Khanna et al.,U.S. Pat. No. 4,351,760. Thus, it is well within the abilities of thoseof skill in the art to choose an energy exchange pair for a particularapplication and to conjugate the members of this pair to a probemolecule, such as, for example, a nucleic acid, peptide or otherpolymer.

As will be apparent to those of skill in the art the methods set forthabove are equally applicable to the coupling to a nucleic acid of groupsother than the fluorescent compounds of the invention, e.g., quenchers,intercalating agents, hybridization enhancing moieties, minor groovebinders, alkylating agents, cleaving agents, etc.

When the nucleic acids are synthesized utilizing an automated nucleicacid synthesizer, the donor and acceptor moieties are preferablyintroduced during automated synthesis. Alternatively, one or more ofthese moieties can be introduced either before or after the automatedsynthesis procedure has commenced. For example, donor and/or acceptorgroups can be introduced at the 3′-terminus using a solid supportmodified with the desired group(s). Additionally, donor and/or acceptorgroups can be introduced at the 5′-terminus by, for example a derivativeof the group that includes a phosphoramidite. In another exemplaryembodiment, one or more of the donor and/or acceptor groups isintroduced after the automated synthesis is complete.

In the dual labeled probes, the quencher moiety is preferably separatedfrom the dye of the invention by at least about 10 nucleotides, and morepreferably by at least about 15 nucleotides. The quencher moiety ispreferably attached to either the 3′- or 5′-terminal nucleotides of theprobe. The dye of the invention moiety is also preferably attached toeither the 3′- or 5′-terminal nucleotides of the probe. More preferably,the donor and acceptor moieties are attached to the 3′- and 5′- or 5′-and 3′-terminal nucleotides of the probe, respectively, althoughinternal placement is also useful.

Once the desired nucleic acid is synthesized, it is preferably cleavedfrom the solid support on which it was synthesized and treated, bymethods known in the art, to remove any protecting groups present (e.g.,60° C., 5 h, concentrated ammonia). In those embodiments in which abase-sensitive group is attached to the nucleic acids (e.g., TAMRA), thedeprotection will preferably use milder conditions (e.g.,butylamine:water 1:3, 8 hours, 70° C.). Deprotection under theseconditions is facilitated by the use of quick deprotect amidites (e.g.,dC-acetyl, dG-dmf).

Peptide Probes

Peptides, proteins and peptide nucleic acids that are labeled with aquencher and a dye of the invention, typically, through an adaptor orlinker-adaptor cassette can be used in both in vivo and in vitroenzymatic assays.

Peptide constructs useful in practicing the invention include those withthe following features: i) a quencher; ii) a dye of the invention; andiii) a cleavage or assembly recognition site for the enzyme. Moreover,the peptide construct is preferably exists in at least one conformationthat allows donor-acceptor energy transfer between the dye of theinvention and the quencher when the fluorophore is excited.

In the dual labeled probes of the invention, the donor and acceptormoieties are connected through an intervening linker moiety. The linkermoiety, preferably, includes a peptide moiety, but can be or can includeanother organic molecular moiety, as well. In a preferred embodiment,the linker moiety includes a cleavage recognition site specific for anenzyme or other cleavage agent of interest. A cleavage site in thelinker moiety is useful because when a tandem construct is mixed withthe cleavage agent, the linker is a substrate for cleavage by thecleavage agent. Rupture of the linker moiety results in separation ofthe dye and the quencher. The separation is measurable as a change indonor-acceptor energy transfer. Alternatively, peptide assembly can bedetected by an increase in donor-acceptor energy transfer between apeptide fragment bearing a fluorescent dye and a peptide fragmentbearing a donor moiety.

When the cleavage agent of interest is a protease, the linker generallyincludes a peptide containing a cleavage recognition sequence for theprotease. A cleavage recognition sequence for a protease is a specificamino acid sequence recognized by the protease during proteolyticcleavage. Many protease cleavage sites are known in the art, and theseand other cleavage sites can be included in the linker moiety. See,e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth.Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994);Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol.244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); Bouvier etal. Meth. Enzymol. 248: 614 (1995), Hardy et al., in AMYLOID PROTEINPRECURSOR IN DEVELOPMENT, AGING, AND ALZHEIMER'S DISEASE, ed. Masters etal. pp. 190-198 (1994).

Solid Support Immobilized Dye Analogues

The amino acid or peptide linked dyes of the invention can beimmobilized on substantially any polymer, biomolecule, or solid orsemi-solid material having any useful configuration. Moreover, anyconjugate comprising one or more dye of the invention can be similarlyimmobilized. In an exemplary embodiment, the dye includes an adaptor ora linker-adaptor casetted and it may be conjugated to the solid supportthrough the adaptor or linker. Alternatively, the dye is attached toanother conjugation component through the adaptor or linker-adaptorcassette. When the support is a solid or semi-solid, examples ofpreferred types of supports for immobilization of the nucleic acid probeinclude, but are not limited to, controlled pore glass, glass plates,polystyrene, avidin coated polystyrene beads, cellulose, nylon,acrylamide gel and activated dextran. These solid supports are preferredbecause of their chemical stability, ease of functionalization andwell-defined surface area. Solid supports such as, controlled pore glass(CPG, 500 Å, 1000 Å) and non-swelling high cross-linked polystyrene(1000 Å) are particularly preferred.

According to the present invention, the surface of a solid support isfunctionalized with a dye of the invention or a species to which a dyeof the invention is conjugated. For clarity of illustration, thefollowing discussion focuses on attaching a reactive dye of theinvention to a solid support. The following discussion is also broadlyrelevant to attaching to a solid support a species that includes withinits structure a dye of the invention.

The dyes of the invention are preferably attached to a solid support byforming a bond between a reactive group on the dye of the invention(e.g., on an amino acid or peptide linker), an adaptor, or alinker-adaptor cassette and a reactive group on the surface of the solidsupport, thereby derivatizing the solid support with one or more dye ofthe invention. Alternatively, the reactive group on the dye of theinvention is coupled with a reactive group on a linker arm attached tothe solid support. The bond between the solid support and the dye of theinvention is preferably a covalent bond, although ionic, dative andother such bonds are useful as well. Reactive groups which can be usedin practicing the present invention are discussed in detail above andinclude, for example, amines, hydroxyl groups, carboxylic acids,carboxylic acid derivatives, alkenes, sulfhydryls, siloxanes, etc.

A large number of solid supports appropriate for practicing the presentinvention are available commercially and include, for example, peptidesynthesis resins, both with and without attached amino acids and/orpeptides (e.g., alkoxybenzyl alcohol resin, aminomethyl resin,aminopolystyrene resin, benzhydrylamine resin, etc. (Bachem)),functionalized controlled pore glass (BioSearch Technologies, Inc.), ionexchange media (Aldrich), functionalized membranes (e.g., —COOHmembranes; Asahi Chemical Co., Asahi Glass Co., and Tokuyama Soda Co.),and the like.

Microarrays

The present invention also provides microarrays including immobilizeddye of the invention and compounds (e.g., peptides, nucleic acids,bioactive agents, etc.) functionalized with a dye of the invention.Moreover, the invention provides methods of interrogating microarraysusing probes that are functionalized with a dye of the invention. Theimmobilized species and the probes are selected from substantially anytype of molecule, including, but not limited to, small molecules,peptides, enzymes nucleic acids and the like.

Nucleic acid microarrays consisting of a multitude of immobilizednucleic acids are revolutionary tools for the generation of genomicinformation, see, Debouck et al., in supplement to Nature Genetics,21:48-50 (1999). The discussion that follows focuses on the use of a dyeof the invention in conjunction with nucleic acid microarrays. Thisfocus is intended to be illustrative and does not limit the scope ofmaterials with which this aspect of the present invention can bepracticed. See, Lehrach, et al., HYBRIDIZATION FINGERPRINTING IN GENOMEMAPPING AND SEQUENCING, GENOME ANALYSIS, Vol. 1, Davies et al, Eds.,Cold Springs Harbor Press, pp. 39-81 (1990), Pirrung et al. (U.S. Pat.No. 5,143,854, issued 1992), and also by Fodor et al., (Science, 251:767-773 (1991), Southern et al. (Genomics, 13: 1008-1017 (1992),Khrapko, et al., DNA Sequence, 1: 375-388 (1991), Kleinfield et al., J.Neurosci. 8:4098-120 (1998)), Kumar et al., Langmuir 10:1498-511 (1994),Xia, Y., J. Am. Chem. Soc. 117:3274-75 (1995), Hickman et al., J. Vac.Sci. Technol. 12:607-16 (1994), Mrkish et al. Ann. Rev. Biophys. Biomol.Struct. 25:55-78 (1996).

Probes of Enzymatic Reactions

In various embodiments, the invention provides a composition which is asubstrate for an enzyme, the substrate comprising a component reactedupon by the enzyme, a fluorescent label component and an amino acid orpeptide linker component conjugating these two components. The adaptorcomponent is of use to control the interaction of the dye with theenzyme.

In various embodiments, the adaptor serves to control the interactionbetween a conjugate of the invention and a protein, such as a DNApolymerase. The adaptor can alter the interaction between the conjugateand the protein through electrostatic, hydrophobic, or stericinteractions. In an exemplary embodiment in which the conjugate isutilized in a single molecule nucleic acid sequencing technique, theadaptor reduces photobleaching of the dye, photodamage to the enzymeand/or the strength of the interaction between the dye and the enzyme.

The Methods

In addition to the compounds of the invention, there is also provided anarray of methods utilizing the compounds. The following discussion isintended to be illustrative of the type and scope of methods with whichthe compounds of the invention can be practiced and should not beinterpreted as being either exhaustive or limiting.

Monitoring Enzymatic Reactions

Peptides, proteins and peptide nucleic acids that are labeled with aquencher and a dye of the invention can be used in both in vivo and invitro enzymatic assays. In an exemplary embodiment, the dye is attachedto the carrier molecule through an adaptor or a linker-adaptor cassette.

Thus, in another aspect, the present invention provides a method fordetermining whether a sample contains an enzyme. The method comprises:(a) contacting the sample with a peptide construct that includes a dyeof the invention; (b) exciting the fluorophore; and (c) determining afluorescence property of the sample, wherein the presence of the enzymein the sample results in a change in the fluorescence property.

Peptide constructs useful in practicing the invention include those withthe following features: i) a quencher; ii) a dye of the invention; andiii) a cleavage or assembly recognition site for the enzyme. Moreover,the peptide construct preferably exists in at least one conformationthat allows donor-acceptor energy transfer between the dye of theinvention and the quencher when the fluorophore is excited.

The assay is useful for determining the presence or amount of enzyme ina sample. For example, by determining the degree of donor-acceptorenergy transfer at a first and second time after contact between theenzyme and the tandem construct, and determining the difference in thedegree of donor-acceptor energy transfer. The difference in the degreeof donor-acceptor energy transfer reflects the amount of enzyme in thesample.

The assay methods also can also be used to determine whether a compoundalters the activity of an enzyme, i.e., screening assays. Thus, in afurther aspect, the invention provides methods of determining the amountof activity of an enzyme in a sample from an organism. The methodincludes: (a) contacting a sample comprising the enzyme and the compoundwith a peptide construct that includes a dye of the invention; (b)exciting the fluorophore; and (c) determining a fluorescence property ofthe sample, wherein the activity of the enzyme in the sample results ina change in the fluorescence property. Peptide constructs useful in thisaspect of the invention are substantially similar to those describedimmediately above.

In a preferred embodiment, the amount of enzyme activity in the sampleis determined as a function of the degree of donor-acceptor energytransfer in the sample and the amount of activity in the sample iscompared with a standard activity for the same amount of the enzyme. Adifference between the amount of enzyme activity in the sample and thestandard activity indicates that the compound alters the activity of theenzyme.

Representative enzymes with which the present invention can be practicedinclude, for example, nucleotide polymerases (e.g., DNA polymerase),trypsin, enterokinase, HIV-1 protease, prohormone convertase,interleukin-1b-converting enzyme, adenovirus endopeptidase,cytomegalovirus assemblin, leishmanolysin, β-secretase for amyloidprecursor protein, thrombin, renin, angiotensin-converting enzyme,cathepsin-D and a kininogenase, and proteases in general.

An exemplary assay for proteases are based on donor-acceptor energytransfer from a donor fluorophore to a quencher placed at opposite endsof a short peptide chain containing the potential cleavage site (see,Knight C. G., Methods in Enzymol. 248:18-34 (1995)). Proteolysisseparates the fluorophore and quencher, resulting in increased intensityin the emission of the donor fluorophore. Existing protease assays useshort peptide substrates incorporating unnatural chromophoric aminoacids, assembled by solid phase peptide synthesis.

In a further aspect, the invention provides a method of monitoring anenzyme reaction. The method generally comprises providing a reactionmixture comprising die enzyme and at least a first reactant composition,the reactant composition comprising a compound having a reactantcomponent, which is a substrate for the enzyme, a fluorescent labelcomponent, and a linker component joining the reactant component to thelabel component. In various embodiments, the linker component increasesthe affinity of the conjugate for the enzyme. In various embodiments,the increased affinity reduces the K_(m) of the reaction, e.g., by 10%,at least 20%, at least 30%, at least 40% or at least 50% relative to theK_(m) of the reaction with an analogous conjugate without the linkercomponent. The reaction mixture is illuminated to excite the fluorescentlabel component, and a fluorescent signal from the reaction mixturecharacteristic of the enzyme reaction is detected.

In an exemplary embodiment, the enzymatic reaction is the reaction of apolymerase with a nucleic acid.

Nucleic Acid Sequencing

In various embodiments, the present invention provides a method fornucleic acid sequencing using one or more compounds of the invention. Anexemplary sequencing method is single molecule nucleic acid sequencing.Exemplary dyes used in sequencing include those in which a nucleic acidis bound to the dye through an adaptor or a dye is bound to a nucleicacid through a linker-adaptor cassette.

Significant interest in the sequencing of single DNA molecules dates to1989 when Keller and colleagues began experimenting with “sequencing bydegradation.” In their experiments, isolated fully-labeled DNA moleculesare degraded by an exonuclease, and individual labeled bases aredetected as they are sequentially cleaved from the DNA (Jett, J. H. etal., J. Biomol. Struct. Dynamics, 7, 301-309 (1989); Stephan, J. et al.,J. Biotechnol., 86, 255-267 (2001); Werner, J. H. et al., J.Biotechnol., 102, 1-14 (2003)). This approach was ultimately compromisedby poor DNA solubility caused by the densely-packed dye labels. Morerecently, alternative single-molecule approaches have been investigated,including “sequencing by synthesis,” where bases are detected one at atime as they are sequentially incorporated into DNA by a polymerase(Braslaysky, I. et al., Proc. Natl. Acad. Sci. USA, 100, 3960-3964(2003); Levene, M. J. et al., Science, 299, 682-686 (2003); Metzker, M.L., Genome Res., 15, 1767-1776 (2005)); and nanopore sequencing whereelectrical signals are detected while single DNA molecules pass throughprotein or solid-state nanopores (Akeson, M. et al., Biophys. J., 77,3227-3233 (1999); Lagerqvist, J. et al., Nano Lett., 6, 779-782 (2006);Rhee, K. J. et al., Annals of emergency medicine, 13, 916-923 (1984)).So far, only sequencing by synthesis has been successful. In the methodof Quake and colleagues (Braslaysky, I. et al., Proc. Natl. Acad. Sci.USA, 100, 3960-3964 (2003)), base-labeled nucleotide triphosphates(dNTPs) are incorporated into DNA immobilized on a microscopecoverglass. Each type of dNTP is applied separately in a fluidics cycle,and incorporated bases are imaged on the surface after washing away theexcess of free nucleotides. While the obtained sequence reads are short,high sequencing rates can potentially be achieved by analyzing billionsof different, individual molecules in parallel with applications inre-sequencing and gene expression profiling.

To obtain long single-molecule reads, potentially tens of kilobases,sequencing-by-synthesis approaches using phosphate-labeled nucleotideshave been developed (Levene, M. J. et al., Science, 299, 682-686(2003)). These nucleotides are labeled with a fluorophore on theterminal phosphate instead of on the base. Labeled nucleotides aredetected while bound to polymerase during the catalytic reaction. Thelabel is released with pyrophosphate as the nucleotide is incorporatedinto DNA. An advantage is that the DNA remains label-free and fullysoluble. Individual polymerase enzymes immobilized on a microscopecoverglass are monitored in real time to detect the sequence ofincorporated nucleotides. In order to achieve long reads, thepolymerase, but not the DNA, can be attached to the coverglass.Polymerase attachment facilitates detection because it keeps the activesite at a single position on the coverglass surface. In the alternativeformat, with the polymerase in solution and the DNA attached, the enzymeactive site would be a moving target for detection, diffusing up toseveral microns from the DNA attachment point as the primer strand isextended from long templates.

U.S. Pat. No. 6,255,083, issued to Williams and incorporated herein byreference, discloses a single molecule sequencing method on a solidsupport. The solid support is optionally housed in a flow chamber havingan inlet and outlet to allow for renewal of reactants that flow past theimmobilized polymerases. The flow chamber can be made of plastic orglass and should either be open or transparent in the plane viewed bythe microscope or optical reader.

Accordingly, it is within the scope of the present invention to utilizethe compounds set forth herein in single molecule DNA sequencing.

In accordance with one embodiment of the methods of invention, thecompounds described herein are used in analyzing nucleic acid sequencesusing a template dependent polymerization reaction to monitor thetemplate dependent incorporation of specific analogs into a synthesizednucleic acid strand, and thus determine the sequence of nucleotidespresent in the template nucleic acid strand. In particular, a polymeraseenzyme is complexed with the template strand in the presence of one ormore nucleotides and/or one or more nucleotide analogs of the invention.In preferred aspects, only the labeled analogs of the invention arepresent representing analogous compounds to each of the four naturalnucleotides, A, T, G and C. When a particular base in the templatestrand is encountered by the polymerase during the polymerizationreaction, it complexes with an available analog that is complementary tosuch nucleotide, and incorporates that analog into the nascent andgrowing nucleic acid strand, cleaving between the a and phosphorus atomsin the analog, and consequently releasing the labeling group (or aportion thereof). The incorporation event is detected, either by virtueof a longer presence of the analog in the complex, or by virtue ofrelease of the label group into the surrounding medium. Where differentlabeling groups are used for each of the types of analogs, e.g., A, T, Gor C, identification of a label of an incorporated analog allowsidentification of that analog and consequently, determination of thecomplementary nucleotide in the template strand being processed at thattime. Sequential reaction and monitoring permits a real-time monitoringof the polymerization reaction and determination of the sequence of thetemplate nucleic acid. As noted above, in particularly preferredaspects, the polymerase enzyme/template complex is provided immobilizedwithin an optical confinement that permits observation of an individualcomplex, e.g., a zero mode waveguide. In addition to their use insequencing, the analogs of the invention are also equally useful in avariety of other genotyping analyses, e.g., SNP genotyping use singlebase extension methods, real time monitoring of amplification, e.g.,RT-PCR methods, and the like. See, for example, U.S. Pat. Nos.7,056,661, 7,052,847, 7,033,764, 7,056,676, 6,917,726, 7,013,054,7,181,122, 7,292,742 and 7,170,050 and 7,302,146, the full disclosuresof which are incorporated herein by reference in their entirety for allpurposes.

The present invention also provides methods of using the compoundsdescribed herein in performing nucleic acid analyses, and particularlynucleic acid sequence analyses. The methods of the invention typicallycomprise providing a template nucleic acid complexed with a polymeraseenzyme in a template dependent polymerization reaction to produce anascent nucleic acid strand, contacting the polymerase and templatenucleic acid with a compound of the invention, and detecting whether ornot a synthon derived from the compound (e.g., monophosphate nucleicacid subunit) was incorporated into the nascent strand during thepolymerization reaction, and identifying a base in the template strandbased upon incorporation of the compound. Preferably, the foregoingprocess is carried out so as to permit observation of individualnucleotide incorporation reactions, through the use of, for example, anoptical confinement, that allows observation of an individual polymeraseenzyme, or through the use of a heterogeneous assay system, where labelgroups released from incorporated analogs are detected.

The invention also provides methods of monitoring nucleic acid synthesisreactions. The methods comprise contacting a polymerase/template/primercomplex with a fluorescently labeled nucleotide or nucleotide analoghaving a nucleotide or nucleotide analog component, a fluorescent labelcomponent, and a linker-adaptor component joining the nucleotide ornucleotide analog component to the label component. A characteristicsignal from the fluorescent dye is then detected that is indicative ofincorporation of the nucleotide or nucleotide analog into a primerextension reaction.

The adaptor linked fluorophores of the invention are of use in singlemolecule or single molecule real time (SMRT) DNA sequencing assays. Ofparticular note in this context is the ability provided by the inventionto design fluorophores with selected absorbance and emission propertiesincluding wavelength and intensity. The compounds of the inventionprovide for very versatile assay design. For example, according to thepresent invention a series of fluorophores of use in an assay arereadily designed to have selected absorbance and emission wavelengthsand emission intensities, allowing multiple fluorophores to be utilizedand distinguished in an assay. In exemplary embodiments, use ofcompounds of the invention in a multifluorophore assay, e.g., singlemolecule DNA sequencing, enhances assay performance by at least about10%, at least about 20% or at least about 30% over a similar assay usingcurrently available fluorophores.

Polymerase Chain Reaction

In another aspect, the invention provides a method for detectingamplification by PCR of a target sequence. Methods of monitoring PCRusing dual labeled nucleic acid probes are known in the art. See, ExpertRev. Mol. Diagn., 5(2), 209-219 (2005). Exemplary dyes used in PCRprobes include those in which a nucleic acid is bound to the dye throughan adaptor or a dye is bound to a nucleic acid through a linker-adaptorcassette.

The dyes and their conjugates described herein can be used insubstantially any nucleic acid probe format for PCR. For example, thedyes of the invention can be incorporated into probe motifs, such asTaqman™ probes (Held et al., Genome Res. 6: 986-994 (1996), Holland etal., Proc. Nat. Acad. Sci. USA 88: 7276-7280 (1991), Lee et al., NucleicAcids Res. 21: 3761-3766 (1993)), molecular beacons (Tyagi et al.,Nature Biotechnology 14:303-308 (1996), Jayasena et al., U.S. Pat. No.5,989,823, issued Nov. 23, 1999)) scorpion probes (Whitcomb et al.,Nature Biotechnology 17: 804-807 (1999)), sunrise probes (Nazarenko etal., Nucleic Acids Res. 25: 2516-2521 (1997)), conformationally assistedprobes (Cook, R., copending and commonly assigned U.S. patentapplication Ser. No. 09/591,185), peptide nucleic acid (PNA)-based lightup probes (Kubista et al., WO 97/45539, December 1997), double-strandspecific DNA dyes (Higuchi et al, Bio/Technology 10: 413-417 (1992),Wittwer et al, BioTechniques 22: 130-138 (1997)) and the like. These andother probe motifs with which the present dyes can be used are reviewedin NONISOTOPIC DNA PROBE TECHNIQUES, Academic Press, Inc. 1992.

Nucleic Acid Detection

In another embodiment, the invention provides a method of detecting atarget nucleic acid in an assay mixture or other sample. The followingdiscussion is generally relevant to the assays described herein. Thisdiscussion is intended to illustrate the invention by reference tocertain preferred embodiments and should not be interpreted as limitingthe scope of probes and assay types in which the compounds of theinvention find use. Other assay formats utilizing the compounds of theinvention will be apparent to those of skill in the art. Exemplary dyesused in sequencing include those in which a nucleic acid is bound to thedye through an adaptor or a dye is bound to a nucleic acid through alinker-adaptor cassette.

An exemplary method uses a dye of the invention or a conjugate thereofto detect a nucleic acid target sequence. The method includes: (a)contacting the target sequence with a detector nucleic acid thatincludes a dye of the invention and a quencher; (b) hybridizing thedetector nucleic acid to the target sequence, thereby altering theconformation of the detector nucleic acid, causing a change in afluorescence parameter; and (c) detecting the change in the fluorescenceparameter, thereby detecting the nucleic acid target sequence.

In various embodiments, the detector nucleic acid includes asingle-stranded target binding sequence. The binding sequence has linkedthereto: i) a quencher; and ii) a dye of the invention. Moreover, priorto its hybridization to a complementary sequence, the detector nucleicacid is preferably in a conformation that allows donor-acceptor energytransfer between the quencher and the dye of the invention when thefluorophore is excited. Furthermore, in the methods described in thissection, a change in fluorescence is detected as an indication of thepresence of the target sequence. The change in fluorescence ispreferably detected in real time.

Kits

In another aspect, the present invention provides kits containing one ormore dye of the invention or a conjugate thereof. In one embodiment, akit includes a reactive dye of the invention and directions forattaching this derivative to another molecule. In another embodiment,the kit includes a dye-labeled polyphosphate nucleic acid in which anadaptor is present between the dye (or dye linker cassette) and thepolyphosphate nucleic acid. The kit further includes one or morecomponent selected from buffers or other compounds or solutions of usein practicing the method, an enzyme (e.g., a DNA polymerase), cofactorsnecessary for enzyme reactions, and directions for performing the assay.

The materials and methods of the present invention are furtherillustrated by the examples that follow. These examples are offered toillustrate, but not to limit the claimed invention.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

EXAMPLES Example 1

1.1 2,3,3-Trimethyl-5-carboxy-3H-indole

A solution of 4-hydrazinobenzoic acid (10.0 g, 65.7 mmol),isopropylmethylketone (21.1 mL, 197 mmol) in acetic acid (35 mL) washeated under reflux in an oil bath for 20 h. After cooling to ambienttemperature the solvent was evaporated off under reduced pressure and toit was added a saturated aqueous solution of NaHCO3 (50 mL) and washedwith CH₂Cl₂ (3×40 mL). The pH of the aqueous solution was adjusted with1 M aqueous HCl to ca. 2, and then extracted with CH₂Cl₂ (3×50 mL). Thecombined organic solution was then dried with Na₂SO₄, filtered andconcentrated to dryness under reduced pressure to yield the desiredproduct (10.7 g, 80%) as a brownish solid.

1.2 2,3,3-Trimethyl-4-carboxy-3H-indole

A solution of 3-hydrazinobenzoic acid (30.0 g, 197 mmol),isopropylmethylketone (31.7 mL, 296 mmol) in ethanol (500 mL) andsulfuric acid (5 mL) was heated under reflux in an oil bath for 20 h.After cooling to ambient temperature the solvent was evaporated offunder reduced pressure to a small volume of ˜200 mL. Collected the solidwith a filter funnel, washed with iPrOH (3×30 mL) and ethyl ether (3×30mL) and dried. Further drying of the solid in an oven at 45° C. underhigh vacuum for 18 h provided 32.59 g (81.3%) of the product.

1.3 2,3,3,-Trimethylindoleninium-5-sulfonate

To an oven dried 500-mL round bottomed flask equipped with a stirringbar, a reflux condenser and a nitrogen balloon was addp-hydrazinobenzenesulfonic acid hemihydrate (50.0 g, 0.253 mol), aceticacid (150 mL), and 3-methyl-2-butanone (84 mL, 0.785 mol). Heated thereaction mixture with stirring in an oil bath at 115° C. for 4 h.Monitored the reaction with TLC (2:1 CH₂Cl₂:MeOH; starting materialR_(f)=0.42, product R_(f)=0.69) until all starting material wasconsumed. Removed oil bath and cooled the reaction solution to ambienttemperature. Slowly added EtOAc (˜200 mL) and the resultant pink solidwere collected via filtration with the aid of EtOAc (2×50 mL). Afterbrief drying the solid was dissolved in MeOH (700 mL). Added KOH (15 g)in iPrOH (200 mL) to the above solution and stirred. Collected theresultant yellow solid via filtration. Washed the solid with iPrOH(2×100 mL), EtOAc (3×100 mL) and air dried. Placed the solid in twoamber bottles and dried in a desiccator under high vacuum overnight.There was obtained 48.8 g (69.5%) of the desired product as a potassiumsalt.

1.4 2,3,3-Trimethyl-4,6-dicarboxy-3H-indole

A solution of 5-hydrazinylbenzene-1,3-dicarboxylic acid (30.0 g, 129mmol), isopropylmethylketone (22.0 mL, 205 mmol) in acetic acid (300 mL)and was heated under reflux in an oil bath for 18 h. After cooling toambient temperature the solvent was evaporated off under reducedpressure to a small volume of ˜100 mL. To the crude mixture was addediPrOH (200 mL) and the solid was collected with a filter funnel, washedwith EtOAc (3×200 mL) and ethyl ether (2×200 mL) and dried. Furtherdrying of the solid in an oven at 50° C. under high vacuum for 18 hprovided 30.44 g (95.5%) of the product.

1.5 2,3,3,-Trimethylindoleninium-4-carboxy-6-sulfonate

To a solid of 2,3,3-trimethyl-4-carboxy-3H-indole (5.20 g, 25.6 mmol)was added oleum (30%, 8 mL, 45.3 mmol) at ambient temperature and heatedto 100° C. for 18 h. After cooling to ambient temperature the acid waspoured into ethyl ether (400 mL). Filtered to collect the solid andwashed the solid with CH3CN (3×50 mL). Re-dissolved the solid in D.I.water (20 mL) and neutralized with KOH to basic (˜pH 10). The aqueoussolvent was evaporated off under reduced pressure to give a solid, whichwas then extracted with MeOH (3×100 mL). After filtering through a padof filter paper the organic extracts were combined and concentrated todryness to give a solid product. Further drying in an oven at 45° C.under high vacuum gave 5.44 g (75.0%) of the off-white solid product.

1.6 2,3,3,-Trimethylindoleninium-5,7-disulfonate and2,3,3,-Trimethylindoleninium-4,6-disulfonate

To a solution of 2,3,3-trimethylindoleninium (2.00 mL, 12.5 mmol) wasadded oleum (30%, 11 mL, 62.3 mmol) and stirred at ambient temperaturefor 48 h followed by heating to 160° C. for 18 h. After cooling toambient temperature the acid was poured into ice water (50 mL) andneutralized with KOH to basic. The aqueous solvent was evaporated offunder reduced pressure to give a solid, which was then extracted withMeOH (3×100 mL). After filtering through a pad of filter paper theorganic extracts were combined and concentrated to dryness. Trituratedthe solid with iPrOH 950 mL) and the solid was collected throughfiltration, washed with EtOAc (3×20 mL) and dried. Further drying in anoven at 45° C. under high vacuum gave a mixture of the titled compound(˜6:4) as an off-white solid (quantitative yield).

1.7 1-(3-Sulfonatopropyl)-2,3,3-trimethylindoleninium

To an oven dried 50-mL round bottom flask equipped with a stir bar,condenser, and an argon balloon in an oil bath was added2,3,3-trimethylindolenine (1.60 mL, 10.0 mmol), 1,3-propanesultone (1.32mL, 15.0 mmol) and 1,2-dichlorobenzene (20 mL). Heated the oil bath to140° C. for 18 h. Removed the oil bath and cooled the mixture to ambienttemperature. Decanted the solvent and triturated the solid with EtOAc(40 mL). Collected the solid using filtration funnel. Re-dissolved thesolid in hot MeOH (20 mL) and concentrated to dryness to give the solidproduct. Further drying in an oven at 45° C. under high vacuum overnightgave 1.96 g (70.0%) of product.

1.8 1-Carboxypentyl-2,3,3-trimethylindoleninium

To an oven dried 250-mL round bottom flask equipped with a stir bar,condenser, and an argon balloon in an oil bath was added2,3,3-trimethylindolenine (5.00 g, 31.4 mmol), bromohexanoic acid (6.74g, 34.5 mmol) and 1,2-dichlorobenzene (250 mL). Heated the oil bath to110° C. for 30 h. Removed the oil bath and cooled the mixture to ambienttemperature. Decanted the solvent and triturated the solid with EtOAc(40 mL). Collected the solid using filtration funnel. Re-dissolved thesolid in MeOH (30 mL) and added EtOAc (100 mL) and Et₂O (400 mL) toprecipitate the solid. The resultant solid was collected, washed with1:1 EtOAc:Hexanes (3×40 mL), Et₂O (3×30 mL) and air dried. Furtherdrying in an oven at 45° C. under high vacuum gave the off-white solid(4.00 g, 36% yield) product. Filtrate still contain lot of product andwas discarded without attempt to isolate more of the product.

1.9 1-(3-Sulfonatopropyl)-2,3,3-trimethylindoleninium-5-carboxylate

A solution of 2,3,3-trimethyl-5-carboxy-3H-indole (193 mg, 0.950 mmol)in 1,3-propanesultone (1 mL) was heated to 145° C. in a sealed tube for20 h. Cooled the tube to ambient temperature and to it was added ethylacetate (20 mL), stirred and the organic solvent was decanted. Repeatedthe process two more times with ethyl acetate (2×20 mL) and the oilyproduct was dried under reduced pressure. Added 6 M HCl (10 mL) to thetube and heated to 60° C. for 4 h. After cooling to ambient temperaturethe solvent was evaporated off under reduced pressure. The crude productwas then purified by reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) to give 300 mg of a solid product (97% yield) afterevaporation of solvent.

1.10 1-(3-Sulfonatopropyl)-2,3,3-trimethylindoleninium-5-sulfonate

To an oven dried 50-mL round bottom flask equipped with a stir bar,condenser, and an argon balloon in an oil bath was added2,3,3-trimethylindoleninium-5-sulfonate (1.50 g, 5.40 mmol),1,3-propanesultone (0.62 mL, 7.0 mmol) and 1,2-dichlorobenzene (15 mL).Heated the oil bath to 140° C. for 48 h. The progress of the reactionwas monitored with analytical HPLC for the disappearance of the startingmaterial, and the formation of the product. Removed oil bath and cooledthe mixture to ambient temperature. Decanted the solvent and trituratedthe solid with EtOAc (40 mL). Collected the solid using filtrationfunnel. Re-dissolved the solid in hot MeOH (40 mL) and added iPrOH (200mL) to precipitate the solid. The resultant solid was collected, washedwith iPrOH (2×50 mL), EtOAc (2×50 mL), ether (2×50 mL) and air dried.The solid was placed in an amber bottle and dried in an oven under highvacuum overnight. There was obtained a total of 1.28 g (59.2%) ofproduct.

1.11 1-Carboxypentyl-2,3,3-trimethylindoleninium-5-sulfonate

To an oven dried 250-mL round bottom flask equipped with a stir bar,condenser, and an argon balloon in an oil bath was added2,3,3-trimethylindolenine-5-sulfonate (10 g, 36.05 mmol), bromohexanoicacid (8.78 g, 45.0 mmol) and 1,2-dichlorobenzene (100 mL). Heated theoil bath to 110° C. for 24 h. Monitored the reaction with TLC (2:1CH₂Cl₂:MeOH) for the disappearance of starting material (R_(f)=0.69) andthe formation of product (R_(f)=0.22). Removed oil bath and cooled themixture to room temperature. Decanted the solvent and triturated thesolid with iPrOH (100 mL). Collected the solid using filtration funnel.Re-dissolved the solid in MeOH (300 mL) and added iPrOH (700 mL) toprecipitate the solid. The resultant solid was collected, washed withiPrOH (2×50 mL), EtOAc (2×50 mL), ether (2×50 mL) and air dried. Thesolid was placed in an amber bottle and dried in a desiccator under highvacuum overnight. There was obtained a total of 8.02 g (62%) of product.

1.12 1-(3-Sulfonatopropyl)-2,3,3-trimethylindoleninium-5,7-disulfonateand 1-(3-Sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-disulfonate

To a 6:4 mixture of 2,3,3,-trimethylindoleninium-5,7-disulfonate and2,3,3,-trimethylindoleninium-4,6-disulfonate (803.2 mg, 2.515 mmol) wasadded 1,3-propanesultone (1.99 mL, 22.6 mmol) in a seal vial. Heated thevial in an oil bath at 140° C. for 72 h. After cooling to ambienttemperature added ethyl acetate (20 mL) to triturate the solid. Decantedthe organic solvent and continue to triturate the solid with EtOAc (3×20mL). Collected the solid using filtration funnel. Re-dissolved the solidin hot MeOH (40 mL) and added iPrOH (200 mL) to precipitate the solid.The resultant solid was collected, washed with iPrOH (2×50 mL), EtOAc(2×50 mL), ether (2×50 mL) and air dried. The solid was placed in anamber bottle and dried in an oven under high vacuum overnight. There wasobtained 660 mg (60%) of product.

1.131-(3-Sulfonatopropyl)-2,3,3-trimethylindoleninium-4-(sulfonatopropylcarboxylate)-6-sulfonate

A mixture of 2,3,3-trimethylindoleninium-4-carboxy-6-sulfonate (218 mg,0.769 mmol) and 1,3-propanesultone (1.5 mL, 17 mmol) in a sealed tubewas heated to 140° C. for 60 h. Cooled the mixture to ambienttemperature and to it was added ethyl acetate (20 mL), stirred and theorganic solvent was decanted. Repeated the process two more times withethyl acetate (2×20 mL) and the oily product was dried under reducedpressure. Added 1 M HCl (15 mL) to the tube and heated to 80° C. for 4h. After cooling to ambient temperature the solvent was evaporated offunder reduced pressure. The crude product was then purified byreverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) to give 263.2 mgof the solid product (64.8% yield).

1.141-(3-Sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-di(sulfonatopropylcarboxylate)

A mixture of 2,3,3-trimethyl-4,6-dicarboxy-3H-indole (600 mg, 2.43mmol), 1,3-propanesultone (5 mL, 57 mmol) and sofolane (3 mL) in asealed tube was heated to 125° C. for 36 h. Cooled the mixture toambient temperature and to it was added ethyl acetate (20 mL), stirredand the organic solvent was decanted. Repeated the process two moretimes with ethyl acetate (2×20 mL) and the oily product was dried underreduced pressure. Added 1 M HCl (10 mL) to the tube and heated to 60° C.for 10 h. After cooling to ambient temperature the solvent wasevaporated off under reduced pressure. The crude product was thenpurified by reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) togive 283 mg (18%) of the solid product and 605 mg (51% yield) of amonoacid ester, presumably the 4-carboxy derivative.

1.152-[(1E)-2-Anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5-carboxylate

A mixture of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-carboxylate (108 mg,0.27 mmol) and N,N′-diphenylformamidine (66 mg, 0.34 mmol) in aceticacid (5 mL) was heated to reflux for 18 h. The progress of the reactionwas monitored with analytical HPLC for the disappearance of startingmaterial, and the formation of product. Extended heating may be neededto completely consume the starting material, however, some of thesymmetrical dye can also be produced. Acetic acid was removed underreduced pressure and the residual dark solid was washed with ethylacetate (3×20 mL). The dried solid was used without further purificationin the next reaction for carbocyanine dye synthesis.

1.162-[(1E)-2-Anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-4-(sulfonatopropylcarboxylate)indoleninium-6-sulfonate

A mixture of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-(sulfonatopropylcarboxylate)-6-sulfonate(50.0 mg, 0.0948 mmol) and N,N′-diphenylformamidine (121 mg, 0.616 mmol)in acetic acid (3 mL) was heated to reflux for 18 h. The progress of thereaction was monitored with analytical HPLC for the disappearance ofstarting material, and the formation of product. Extended heating may beneeded to completely consume the starting material, however, some of thesymmetrical dye can also be produced. Acetic acid was removed underreduced pressure and the residual dark solid was washed with ethylacetate (3×20 mL), dried and purified by reverse-phase HPLC(acetonitrile/0.1 M TEAB gradient) to give 41 mg of the solid product(70% yield).

1.172-[(1E)-2-Anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5,7-disulfonateand2-[(1E)-2-Anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4,6-disulfonate

A mixture of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5,7-disulfonate and1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-disulfonate (˜6:4,161.2 mg, 0.366 mmol) and N,N′-diphenylformamidine (86 mg, 0.44 mmol) inacetic acid (2 mL) and acetic anhydride (2 mL) was heated to reflux for18 h. The progress of the reaction was monitored with analytical HPLCfor the disappearance of the starting material, and the formation of theproduct. Extended heating may be needed to completely consume thestarting material, however, some of the symmetrical dye can also beproduced. Solvent was removed under reduced pressure and the residualdark solid was washed with ethyl acetate (3×20 mL). The dried solid wasused without further purification in the next reaction for carbocyaninedye synthesis.

1.182-[(1E,3E)-4-Anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5-carboxylate

A mixture of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-carboxylate (300 mg,0.922 mmol) and malonaldehyde dianil hydrochloride (310 mg, 1.20 mmol)in acetic acid (10 mL) was heated to reflux for 16 h. The progress ofthe reaction was monitored with analytical HPLC for the disappearance ofthe starting material, and the formation of the product. Acetic acid wasremoved under reduced pressure and the residual dark solid was washedwith ethyl acetate (3×20 mL). The dried solid was used without furtherpurification in the next reaction for dicarbocyanine dye synthesis.

1.192-[(1E,3E)-4-Anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4-(sulfonatopropylcarboxylate)-6-sulfonate

A mixture of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-(sulfonatopropylcarboxylate)-6-sulfonate(50.0 mg, 0.0948 mmol) and malonaldehyde dianil hydrochloride (48.0 mg,0.185 mmol) in acetic acid (3 mL) was heated to reflux for 18 h. Theprogress of the reaction was monitored with analytical HPLC for thedisappearance of the starting material, and the formation of theproduct. Acetic acid was removed under reduced pressure and the residualdark solid was washed with ethyl acetate (3×20 mL), dried and purifiedby reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) to give 35 mgof the solid product (56.3% yield).

1.202-[(1E,3E)-4-Anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4-carboxy-6-sulfonate

A solution of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-carboxy-6-sulfonate(72.9 mg, 0.180 mmol) and malonaldehyde dianil hydrochloride (56.0 mg,0.216 mmol) in acetic anhydride (2 mL) and acetic acid (3 mL) was heatedto reflux for 26 h. The progress of the reaction was monitored withanalytical HPLC for the disappearance of the starting material, and theformation of the product. Solvent was removed under reduced pressure andthe residual dark solid was washed with ethyl acetate (3×20 mL). Thedried solid was used without further purification in the next reactionfor dicarbocyanine dye synthesis.

1.212-[(1E,3E)-4-Anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5,7-disulfonateand2-[(1E,3E)-4-Anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4,6-disulfonate

A mixture of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5,7-disulfonate and1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-disulfonate (˜6:4,195 mg, 0.442 mmol) and malonaldehyde dianil hydrochloride (126 mg,0.487 mmol) in acetic acid (2.4 mL) and acetic anhydride (2.4 mL) washeated to reflux for 4 h. Solvent was removed under reduced pressure andthe residual dark solid was washed with ethyl acetate (3×20 mL). Thedried solid was used without further purification in the next reactionfor dicarbocyanine dye synthesis.

1.22 Preparation of 1.22

To a solution of2-[(1E)-2-anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5-carboxylate(37.7 mg, 0.088 mmol) and1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-sulfonate (38.7 mg,0.097 mmol) in N,N-dimethylformamide (5 mL) was added Ac₂O (2 mL),pyridine (2 mL) and stirred at ambient temperature for 24 h. Solvent wasevaporated off under reduced pressure to give a dark red residue, whichwas then purified by reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) to give 41.7 umole of the product (47% yield). λmax (553 nm).

1.23 Preparation of 1.23

A solution of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-carboxylate (473 mg,1.45 mmol) and triethyl orthoformate (166 uL, 1.6 mmol) in pyridine (5mL) was heated to reflux for 2 h. Solvent was evaporated off underreduced pressure to give a dark red residue, which was then purified byreverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) to give 0.181 mmolof the product (25% yield). λmax (556 nm).

1.24 Preparation of 1.24

A solution of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-sulfonate (46.6 mg,0.117 mmol), 1-carboxypentyl-2,3,3-trimethylindoleninium-5-sulfonate(41.2 mg, 0.117 mmol) and triethyl orthoformate (20 uL, 0.12 mmol) inpyridine (3 mL) was heated to reflux for 48 h. Solvent was evaporatedoff under reduced pressure to give a dark red residue, which was thenpurified by reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) togive 1.71 umol of the product (3% yield). λmax (551 nm)

1.25 Preparation of 1.25

A solution of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-sulfonate (97.9 mg,0.245 mmol) and N,N′-diphenylformamidine (24.0 mg, 0.122 mmol) in aceticanhydride (3 mL) and pyridine (5 mL) was heated to reflux for 18 h.Solvent was evaporated off under reduced pressure to give a dark redresidue, which was then purified by reverse-phase HPLC (acetonitrile/0.1M TEAB gradient) to give the product. λmax (550 nm).

1.26 Preparation of 1.26B and 1.26C

A solution of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-sulfonate (85.5 mg,0.214 mmol), 1-carboxypentyl-2,3,3-trimethylindolenine (75.8 mg, 0.214mmol) and triethyl orthoformate (40 uL, 0.24 mmol) in pyridine (3 mL)and MeOH (3 mL) was heated to reflux for 24 h. Solvent was evaporatedoff under reduced pressure to give a dark red residue, which was thenpurified by reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) togive 3 carbocyanine dyes, 1.26A ((λmax, 550 nm), 1.26B (λmax, 549 nm)and diacid symmetrical dye 1.26C (λmax, 549 nm).

1.27 Preparation of 1.27B and 1.27C

A solution of 1,2,3,3-tetramethylindoleninium (79.0 mg, 0.262 mmol),1-carboxypentyl-2,3,3-trimethylindoleninium (92.9 mg, 0.262 mmol) andtriethyl orthoformate (45 uL, 0.27 mmol) in pyridine (3 mL) and MeOH (3mL) was heated to reflux for 2 h. Additional triethyl orthoformate (45uL, 0.27 mmol) was added and continue to reflux for 3 h. Solvent wasevaporated off under reduced pressure to give a dark red residue, whichwas then purified by reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) to give 3 carbocyanine dyes, 1.27A (λmax, 549 nm), 1.27B(λmax, 545 nm) and dimethyl symmetrical dye 1.27C (λmax, 543 nm).

1.28 Preparation of 1.28A and 1.28B

A solution of 1,2,3,3-tetramethylindoleninium (91.3 mg, 0.303 mmol),1-carboxypentyl-2,3,3-trimethylindoleninium-5-sulfonate (107.1 mg, 0.303mmol) and triethyl orthoformate (55 uL, 0.33 mmol) in pyridine (3 mL)and MeOH (1 mL) was heated to reflux for 2 h. Additional triethylorthoformate (55 uL, 0.33 mmol) was added and continue to reflux for 3h. Solvent was evaporated off under reduced pressure to give a dark redresidue, which was then purified by reverse-phase HPLC (acetonitrile/0.1M TEAB gradient) to give 3 carbocyanine dyes, 1.28A (λmax, 553 nm),1.28B (λmax, 548 nm) and dimethyl symmetrical dye 1.28C (λmax, 543 nm).

1.29 Preparation of 1.29A and 1.29B

A solution of2-[(1E)-2-anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5,7-disulfonateand2-[(1E)-2-anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4,6-disulfonate(˜6:4, 66 mg, 0.122 mmol),1-carboxypentyl-2,3,3-trimethylindoleninium-5-sulfonate (43.1 mg, 0.122mmol) in N,N-dimethylformamide (2 mL) and acetic anhydride (1 mL) andpyridine (1 mL) was stirred a ambient temperature for 24 h. Solvent wasevaporated off under reduced pressure to give a dark red residue, whichwas then purified by reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) to give 1.8 umol of the two products in ˜6:4. λmax (560 nm,1.29A); λmax (558 nm, 1.29B).

1.30 Preparation of 1.30A and 1.30B

A solution of2-[(1E)-2-anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5,7-disulfonateand2-[(1E)-2-anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4,6-disulfonate(˜6:4, 148 mg, 0.272 mmol),1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-carboxylate (70.0mg, 0.215 mmol) in N,N-dimethylformamide (3 mL) and acetic anhydride (1mL) and pyridine (1 mL) was stirred at ambient temperature for 24 h.Solvent was evaporated off under reduced pressure to give a dark redresidue, which was then purified by reverse-phase HPLC (acetonitrile/0.1M TEAB gradient) to give 2.1 umol of the two products in ˜6:4. λmax (554nm, 1.30A); λmax (550 nm, 1.30B).

1.31 Preparation of 1.31

A solution of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-(sulfonatopropylcarboxylate)-6-sulfonate(25 mg, 0.062 mmol) and triethyl orthoformate (30 uL, 0.18 mmol) inpyridine (2 mL) was heated to reflux inside a sealed vial for 2.5 h.Solvent was evaporated off under reduced pressure to give a dark redresidue, which was then purified by reverse-phase HPLC (acetonitrile/0.1M TEAB gradient) to give 0.0154 mmol of the product (25% yield). λmax(540 nm).

1.32 Preparation of 1.32A and 1.32B

A solution of 1.31 (35 umol) in 6N HCl (4 mL) was heated in a sealedvial for 5. After cooling to ambient temperature the solvent wasevaporated off under reduced pressure and residual solid was purified byreverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) to give 1.32A(22.6 umol, λmax 543 nm, 64.5% yield), 1.32B (11.2 umol, λmax 543 nm,32% yield) and a small amount of starting material.

1.33 Preparation of 1.33

To a solution of2-[(1E)-2-anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-4-(sulfonatopropylcarboxylate)indoleninium-6-sulfonate(3.6 mg, 7.1 umol),1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-di(sulfonatoethylcarboxyamide)(5 mg, 8.5 umol) in N,N-dimethyl formamide (2 mL) was added acteicanhydride (50 uL) and triethylamine (50 uL) and stirred at ambienttemperature for 18 h. Solvent was evaporated off under reduced pressureto give a dark red residue, which was then purified by reverse-phaseHPLC (acetonitrile/0.1 M TEAB gradient) to give 0.19 umol of the product(3% yield). λmax (543 nm).

1.34 Preparation of 1.34B

A solution of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-di(sulfonatoethylcarboxyamide)(12 mg, 30 umol),1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-carboxy-6-sulfonate(10 mg, 17 umol) and triethyl orthoformate (30 uL, 0.18 mmol) inpyridine (1 mL) was heated to reflux for 2 h. Solvent was evaporated offunder reduced pressure to give a dark red residue, which was thenpurified by reverse-phase HPLC (acetonitrile/0.1 M TEAB gradient) togive 3 carbocyanine dyes, 1.34A (λmax, 543 nm), 1.34B (λmax, 544 nm) anda small amount of the tetraamide symmetrical dye.

1.35 Preparation of 1.35B and 1.35C

A solution of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-di(sulfonatopropylcarboxylate)(25.6 mg, 41.7 umol),1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-carboxy-6-sulfonate(16.9 mg, 41.7 umol), KOAc (12.5 mg) and triethyl orthoformate (30 uL,0.18 mmol) in H₂O (250 uL) and MeOH (1 mL) was heated to reflux for 2 h.Solvent was evaporated off under reduced pressure to give a dark redresidue, which was then purified by reverse-phase HPLC (acetonitrile/0.1M TEAB gradient) to give 3 carbocyanine dyes, 1.35A (λmax, 543 nm),1.35B (λmax, 542 nm) and the tetra-ester symmetrical dye 1.35C (λmax,540 nm).

1.36 Preparation of 1.36A and 1.36B

A solution of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5,7-disulfonateand2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4,6-disulfonate(˜6:4, 126 mg, 0.220 mmol),1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-carboxylate (71.6mg, 0.220 mmol) in N,N-dimethylformamide (3 mL) and acetic anhydride (1mL) and pyridine (1 mL) was stirred at ambient temperature for 24 h.Solvent was evaporated off under reduced pressure to give a dark blueresidue, which was then purified by reverse-phase HPLC (acetonitrile/0.1M TEAB gradient) to give 1.36A (λmax 658 nm) and 1.36B (λmax 653 nm).

1.37 Preparation of 1.37A and 1.37B

A solution of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5,7-disulfonateand2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4,6-disulfonate(˜6:4, 126 mg, 0.220 mmol),1-carboxypentyl-2,3,3-trimethylindoleninium-5-sulfonate (77.8 mg, 0.220mmol) in N,N-dimethylformamide (3 mL) and acetic anhydride (1 mL) andpyridine (1 mL) was stirred at ambient temperature for 24 h. Solvent wasevaporated off under reduced pressure to give a dark blue residue, whichwas then purified by reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) to give 1.37A (λmax 653 nm) and 1.37B (λmax 651 nm).

1.38 Preparation of 1.38A and 1.38B

A solution of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5,7-disulfonate and2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4,6-disulfonate (˜6:4, 161 mg, 0.282 mmol),1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-carboxylate (70.0mg, 0.214 mmol) in N,N-dimethylformamide (3 mL) and acetic anhydride (1mL) and pyridine (1 mL) was stirred at ambient temperature for 24 h.Solvent was evaporated off under reduced pressure to give a dark blueresidue, which was then purified by reverse-phase HPLC (acetonitrile/0.1M TEAB gradient) to give 8.4 umol of a mixture of 1.38A (λmax 650 nm)and 1.38B (λmax 648 nm).

1.39 Preparation of 1.39

A solution of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-(sulfonatopropylcarboxylate)-6-sulfonate(25 mg, 0.062 mmol) and 1,1,3,3-tetraethoxypropane (40 uL, 0.243 mmol)in pyridine (2 mL) was heated to reflux inside a sealed vial for 2 h.Solvent was evaporated off under reduced pressure to give a dark blueresidue, which was then purified by reverse-phase HPLC (acetonitrile/0.1M TEAB gradient) to give the product (λmax 640 nm).

1.40 Preparation of 1.40

To a solution of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4-(sulfonatopropylcarboxylate)-6-sulfonate(6.3 mg, 12 umol) and1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-di(sulfonatoethyllcarboxyamide(7.6 mg, 13 umol) in N,N-dimethylformamide (2 mL) was added aceticanhydride (50 uL), triethylamine (50 uL) and stirred at ambienttemperature for 18 h. Solvent was evaporated off under reduced pressureto give a dark blue residue, which was then purified by reverse-phaseHPLC (acetonitrile/0.1 M TEAB gradient) to give 3 umol of the product(25% yield). λmax (640 nm).

1.41 Preparation of 1.41A and 1.41B

To a solution of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4-carboxy-6-sulfonate(9.3 mg, 16 umol), a 6:4 mixture of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5,7-di sulfonate and1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-disulfonate (8.5mg, 19 umol) in N,N-dimethylformamide (1 mL) was added acetic anhydride(100 uL) and triethylamine (100 uL) and stirred at ambient temperaturefor 18 h. Solvent was evaporated off under reduced pressure to give adark blue residue, which was then purified by reverse-phase HPLC(acetonitrile/0.1 M TEAB gradient) to give a mixture of the product(8.5%), 1.41A (λmax 650 nm) and 1.41B (λmax 648 nm).

1.42 Preparation of 1.42

To a solution of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4-carboxy-6-sulfonate(26.6 mg, 46 umol),1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-di(sulfonatoethyllcarboxyamide(27 mg, 46 umol) in N,N-dimethylformamide (1 mL) was added aceticanhydride (100 uL) and triethylamine (100 uL) and stirred at ambienttemperature for 18 h. Solvent was evaporated off under reduced pressureto give a dark blue residue, which was then purified by reverse-phaseHPLC (acetonitrile/0.1 M TEAB gradient) to give the product (9.6 umol,21%), (λmax 641 nm).

1.43 Preparation of 1.43

To a solution of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4-carboxy-6-sulfonate(11 mg, 19 umol),1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-(sulfonatopropylcarboxylate)-6-sulfonate(10 mg, 19 umol) in N,N-dimethylformamide (1 mL) was added aceticanhydride (100 uL) and triethylamine (100 uL) and stirred at ambienttemperature for 18 h. Solvent was evaporated off under reduced pressureto give a dark blue residue, which was then purified by reverse-phaseHPLC (acetonitrile/0.1 M TEAB gradient) to give the product (3.8 umol,20%), (λmax 640 nm).

1.44 Preparation of 1.44

To a solution of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4-carboxy-6-sulfonate(6.4 mg, 10 umol),1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-di(sulfonatopropylcarboxylate)(6.0 mg, 10 umol) in N,N-dimethylformamide (1 mL) was added aceticanhydride (100 uL) and triethylamine (100 uL) and stirred at ambienttemperature for 18 h. Solvent was evaporated off under reduced pressureto give a dark blue residue, which was then purified by reverse-phaseHPLC (acetonitrile/0.1 M TEAB gradient) to give the product (1.9 umol,19%), (λmax 640 nm).

1.45 Preparation of 1.45

A solution of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-carboxy-6-sulfonate(65.2 mg, 0.161 mmol) and 1,1,3,3-tetraethoxypropane (100 uL, 0.61 mmol)in pyridine (2 mL) was heated to reflux inside a sealed vial for 2 h.Solvent was evaporated off under reduced pressure to give a dark blueresidue, which was then purified by reverse-phase HPLC (acetonitrile/0.1M TEAB gradient) to give 15.4 umol (10%) the product (λmax 641 nm).

1.46 Preparation of 1.46

To a solution of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4-carboxylate(11.2 mg, 24.6 umol),1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4,6-di(sulfonatopropylcarboxylate)(12.0 mg, 19.5 umol) in N,N-dimethylformamide (0.5 mL) was added aceticanhydride (40 uL) and triethylamine (40 uL) and stirred at ambienttemperature for 18 h. Solvent was evaporated off under reduced pressureto give a dark blue residue, which was then purified by reverse-phaseHPLC (acetonitrile/0.1 M TEAB gradient) to give the product (6.79 umol,34.8%), (λmax 638 nm).

1.47 Preparation of 1.47

To a solution of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-4-carboxylate(8.6 mg, 19 umol),1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-4-sulfonatopropylcarboxylate-6-sulfonate(10.0 mg, 18.9 umol) in N,N-dimethylformamide (0.5 mL) was added aceticanhydride (40 uL) and triethylamine (40 uL) and stirred at ambienttemperature for 18 h. Solvent was evaporated off under reduced pressureto give a dark blue residue, which was then purified by reverse-phaseHPLC (acetonitrile/0.1 M TEAB gradient) to give the product (4.3 umol,23%), (λmax 637 nm).

1.48 4-Methyl-5-oxo-1-hexanesulfonate

To a solution of ethyl 2-methylacetoacetate (30.0 mL, 208 mmol) wasadded 1M t-BuOK in t-BuOH (229 mL, 229 mmol) and 1,3-propanesultone(20.2 mL, 229 mmol) and heated in an oil bath to reflux for 24 h. Aftercooling to ambient temperature a solid was formed which was trituratedwith EtOAc (200 mL) and the resultant solid was collected throughfiltration, washed with ethyl acetate (2×20 mL) and dried. To the driedsolid was added 50% HCl (100 mL) and the solution was heated at 110° C.for 24 h. Solvent was evaporated off under reduced pressure andco-evaporated with acetonitrile (2×30 mL) to give 38.3 g (95%) of anoily product. The crude residue was used without further purification.

1.49 2,3-Dimethyl-3-(3-sulfopropyl)indoleninium-5-sulfonate

To an oven dried 100-mL round bottomed flask equipped with a stirringbar, a reflux condenser and a nitrogen balloon was addp-hydrazinobenzenesulfonic acid hemihydrate (2.0 g, 0.010 mol), aceticacid (25 mL), and 4-methyl-5-oxo-1-hexanesulfonate (5.5 g, 0.028 mol).Heated the reaction mixture to reflux with stirring in an oil bath at115° C. for 30 h. Removed the oil bath and cooled the reaction solutionto ambient temperature. Solvent was evaporated off under reducedpressure to dryness. The residual crude product was triturated withMeOH/iPrOH (1:5, 100 mL) and filtered. The solid was then dissolved inMeOH (400 mL) and filtered to remove the undissolved solid (startingmaterial). The filtrate was poured into a beaker and to it was added KOH(1.19 g, 0.021 mol) in iPrOH (400 mL) and stirred. The resultant solidwas collected, washed with iPrOH (2×20 mL), EtOAc (2×20 mL) and dried.Placed the solid in an amber bottle and dried in an oven at 50° C. underhigh vacuum for 18 h. There was obtained 2.94 g (68.4%) of the desiredproduct as a potassium salt.

1.50 2,3-Dimethyl-1,3-bis(3-sulfonatopropyl)indoleninium-5-sulfonate

To an oven dried 50-mL round bottom flask equipped with a stir bar,condenser, and an argon balloon in an oil bath was added2,3-dimethyl-3-(3-sulfopropyl)indoleninium-5-sulfonate (530 mg, 1.25mmol), 1,3-propanesultone (0.554 mL, 626 mmol) and 1,2-dichlorobenzene(5 mL). Heated the oil bath to 135° C. for 24 h. Removed oil bath andcooled the mixture to ambient temperature. Decanted the solvent andtriturated the solid with iPrOH (40 mL). The resultant solid wascollected, washed with iPrOH (2×50 mL), EtOAc (2×50 mL), ether (2×50 mL)and air dried. The solid was placed in an amber bottle and dried in anoven under high vacuum overnight. The solid product was used withoutfurther purification.

Example 2

2.1 Synthesis of 2b

TSTU (3.76 g, 12.54 mmole), in 4 portions, was added to a solution of4-bromo-2,6-pyridinedicarboxylic acid (2a, 1.03 g, 4.12 mmole) anddiisopropylethylamine (1.68 g, 13.0 mmole) in 20 mL DMF at 4° C. over 3min. The reaction was stirred for 10 min, the cooling bath was removed,and stirred for another 1 hr. This DMF solution was added to a solutionof taurine (12.0 g, 95.8 mmole) and Na₂CO₃ (16.2 g, 152.8 mmole) in 300mL of DMF/H₂O (1:10) over 20 min at room temperature. The reaction wasstirred for another 30 min after the addition was completed. The productwas purified by reverse phase HPLC eluted with a gradient of CH₃CN over0.1 N triethylammonium bicarbonate (TEAB) buffer (pH 7). The yield ofthe desired product (2b) was 1.09 g, also isolated was the mono-taurineamide 0.58 g.

2.2 Synthesis of 2d

A solution of Na₂CO₃ (214 mg, 2 mmole) in 2.5 mL H₂O was added dropwiseto a solution of 2b (270 mg, 0.40 mmole) and 2c (100 mg, 0.43 mmole) in5 mL DMF with stirring. Argon was bubbled through the result mixture for1 hr, Pd(PPh₃)₄ (29 mg, 0.025 mmole) was added to the mixture afterbubbling for 30 min. The result mixture was heated at 100° C. for 4 hrunder a slight positive pressure of argon. After cooling down, thesolvents were removed in vacuo, the residue was redissolved in 10 mL 0.1N TEAB buffer, filtered and purified by reverse phase HPLC eluted with agradient of CH₃CN over 0.1 N TEAB buffer. The yield of the desiredproduct (2.2) was 220 mg.

2.3 Synthesis of 2e

A solution NaNO₂ (33 mg, 0.47 mmole) in 0.5 mL H₂O was added to asolution of 2d (220 mg, 0.32 mmole) in 6 mL 4 N HCl at −5˜−10° C. over 5min. During the course of addition the temperature was kept below −5° C.The reaction was stirred for another 15 min after the addition wascompleted. SnCl₂.2H₂O (300 mg, 1.33 mmole) in 1 mL 6N HCl, pre-cooled at−5° C., was added dropwise to the reaction at −5° C. over 5 min. Thereaction was stirred for 4 hr as the cooling bath gradually warmed toroom temperature. The reaction was diluted with 10 mL H₂O, filtered,neutralized with 1 M NaOH to pH 9, and purified by reverse phase HPLCeluted with a gradient of CH₃CN over 0.1 N TEAB buffer. The yield of theproduct (DS374-28) was 172 mg.

2.4 Synthesis of 2f

A solution of 2e (172 mg, 0.25 mmole) and methyl isopropyl ketone (130mg, 1.5 mmole) in 5 mL of HOAc was heated at 120° C. for 6 hr. HOAc wasremoved in vacuo, the residue redissolved in 0.1 N TEAB, filtered, andpurified by reverse phase HPLC eluted with a gradient of CH3CN over 0.1N TEAB buffer. The yield of the product was 101 mg.

2.5 Synthesis of 2g

2f (40 mg, 0.054 mmole) dissolved in 0.5 mL of sulfolane at 80° C. wasadded 1,3-Propanesultone (67 mg, 0.54 mmole) with stirring. The reactionwas heated to 120° C. for 1.5 hr. After cooling down the reaction wasdiluted with 10 mL 0.1 N TEAB, filtered, and purified by reverse phaseHPLC eluted with a gradient of CH₃CN over 0.1 N TEAB buffer. The yieldof the product was 36 mg.

2.6 Synthesis of 2g′

2f (60 mg, 0.081 mmole) dissolved in 0.6 mL of sulfolane at 80° C. wasadded 6-bromohexanoic acid (222 mg, 1.13 mmole) with stirring. Thereaction was heated to 130° C. for 3 hr. After cooling down the reactionwas diluted with 10 mL 0.1 N TEAB, filtered, and purified by reversephase HPLC eluted with a gradient of CH3CN over 0.1 N TEAB buffer. Theyield of the product was 33 mg.

2.7. Synthesis of 2h and 2h′

2g (10 mg, 0.01 mmole) and 2g′ (10 mg, 0.01 mmole) dissolved in 0.3 mLof sulfolane and 0.2 mL of pyridine at 80° C. was added 1,1,3,3tetramethoxypropane (8.2 mg, 0.05 mmole) with stirring. The reaction washeated to 120° C. for 3 hr, during which time additional 1,1,3,3tetramethoxypropane (8.2 mg, 0.05 mmole) was added to the reaction.After cooling down the reaction was diluted with 10 mL of 0.1 N TEAB,filtered, and purified by reverse phase HPLC eluted with a gradient ofCH3CN over 0.1 N TEAB buffer. The yield of the product was 1.9 mg. Alsoisolated from the reaction was 2h′ in 3.1 mg.

2.8 Synthesis of 2j

A solution of Na₂CO₃ (144 mg, 1.36 mmole) in 2.5 mL H₂O was addeddropwise to a solution of 2b (175 mg, 0.27 mmole) and 2i (75 mg, 0.30mmole) in 5 mL of DMF with stirring. Argon was bubbled through theresult mixture for 1 hr, Pd(PPh₃)₄ (17 mg, 0.015 mmole) was added to themixture after bubbling for 30 min. The result mixture was heated at 100°C. for 4 hr under a slight positive pressure of argon. The solvents wereremoved in vacuo, the residue was redissolved in 10 mL 0.1 N TEABbuffer, filtered and purified by reverse phase HPLC eluted with agradient of CH₃CN over 0.1 N TEAB buffer. The yield of the desiredproduct was 137 mg.

2.9 Synthesis of 2k

A solution NaNO₂ (27 mg, 0.39 mmole) in 0.5 mL H₂O was added to asolution of 2j (137 mg, 0.19 mmole) in 3 mL 4 N HCl at −5˜−10° C. over 2min. During the course of addition the temperature was kept below −5° C.The reaction was stirred for another 30 min after the addition wascompleted. SnCl₂.2H₂O (210 mg, 0.93 mmole) in 0.5 mL 6N HCl, pre-cooledat −5° C., was added dropwise to the reaction at 5° C. over 2 min. Thereaction was stirred for 4 hr as the cooling bath gradually warmed toroom temperature. The reaction was diluted with 10 mL of H₂O, filtered,neutralized with 1 M NaOH to pH 9, and purified by reverse phase HPLCeluted with a gradient of CH3CN over 0.1 N TEAB buffer. The yield of theproduct was 88 mg.

2.10 Synthesis of 21

A solution of 2k (78 mg, 0.12 mmole) and methyl isopropyl ketone (104mg, 1.2 mmole) in 3 mL HOAc was heated at 50° C. for 0.5 hr. HOAc wasremoved in vacuo, the residue redissolved in 0.1 N TEAB, filtered, andpurified by reverse phase HPLC eluted with a gradient of CH₃CN over 0.1N TEAB buffer. The yield of the product was 46 mg.

2.11 Synthesis of 2m

2l (22 mg, 0.028 mmole) dissolved in 0.25 mL of sulfolane at 80° C. wasadded 1,3-Propanesultone (46 mg, 0.37 mmole) with stirring. The reactionwas heated to 120° C. for 1.5 hr. After cooling down the reaction wasdiluted with 10 mL 0.1 N TEAB, filtered, and purified by reverse phaseHPLC eluted with a gradient of CH₃CN over 0.1 N TEAB buffer. The yieldof the product was 15 mg.

2.12 Synthesis of 2n

2l (50 mg, 0.065 mmole) dissolved in 0.5 mL of sulfolane at 80° C. wasadded 6-bromohexanoic acid (108 mg, 0.55 mmole) with stirring. Thereaction was heated to 130° C. for 3 hr. After cooling down the reactionwas diluted with 10 mL 0.1 N TEAB, filtered, and purified by reversephase HPLC eluted with a gradient of CH₃CN over 0.1 N TEAB buffer. Theyield of the product was 26 mg.

2.13 Synthesis of 2o and 2o′

2m (15 mg, 0.015 mmole) and 2n (15 mg, 0.015 mmole) dissolved in 0.5 mLof sulfolane and 0.3 mL of pyridine at 80° C. was added 1,1,3,3tetramethoxypropane (8.2 mg, 0.05 mmole) with stirring. The reaction washeated to 120° C. for 3 hr, during which time additional 1,1,3,3tetramethoxypropane (8.2 mg, 0.05 mmole) was added to the reaction.After cooling down the reaction was diluted with 10 mL 0.1 N TEAB,filtered, and purified by reverse phase HPLC eluted with a gradient ofCH₃CN over 0.1 N TEAB buffer. The yield of the product (2o′) was 3.2 mg.Also isolated from the reaction was 2o (3.3 mg).

2.14 Synthesis of 2q

2p (500 mg, 2.02 mmole) and 3 mL of 1,3 propanesultone was heated at120° C. for 3 hr. After cooling down, 15 mL of 6 N HCl was slowly addedto the reaction and heated to 100° C. for 5 hr. The reaction was dilutedwith 15 mL of CH₃CN and filtered. The filtrate was concentrated in vacuoand resuspended in 50 mL of EtOH. The precipitated was collected, washedwith EtOH, Et₂O and dried under vacuum. The yield of the product was 614mg.

2.15 Synthesis of 2r

2q (20 mg, 0.054 mmole) and diphenyl formamidine (13 mg, 0.064 mmole)was heated in 0.5 mL of HOAc at 120° C. for 2 hr. After cooling down,the reaction was diluted with 5 mL of EtOAc. The precipitate wascollected, washed with EtOAc, dried under vacuum, and redissolved in 0.5mL of DMF. 2q (20 mg, 0.054 mmole) was added to the DMF solutionfollowed by Et₃N (28 mg, 0.27 mmole), Ac₂O (28 mg, 0.27 mmole), and thereaction was stirred for 3 hr at room temperature. The reaction wasdiluted with 10 mL 0.1 N TEAB, filtered, and purified by reverse phaseHPLC eluted with a gradient of CH₃CN over 0.1 N TEAB buffer. The yieldof the product was 10.5 mg.

2.16 Synthesis of 2s

7 (potassium salt, 180 mg, 0.46 mmole) and diphenyl formamidine (100 mg,0.51 mmole) was heated in 4 mL of HOAc at 120° C. for 5 hr. During whichtime more diphenyl formamidine (50 mg, 0.25 mmole) was added to thereaction. After cooling down, the reaction was diluted with 30 mL ofEtOAc, the precipitate was collected, washed with EtOAc, Et₂O, and driedunder vacuum. The yield of the crude product was 215 mg and was usedwithout further purification.

2.17 Synthesis of 2t

To a stirring solution of 2q (50 mg, 0.13 mmole) and 2s (75 mg, crude)in 3 mL of DMF was added Et₃N (66 mg, 0.65 mmole) followed by Ac₂O (66mg, 0.65 mmole). The reaction was stirred at room temperature for 3 hrand quenched with 12 mL EtOAc. The precipitated was collected andpurified by reverse phase HPLC eluted with a gradient of CH₃CN over 0.1N TEAB buffer. The yield of the product was 24 mg.

2.18 Synthesis of 2v

2u (267 mg, 0.40 mmole) dissolved in 1 mL of sulfolane at 80° C. wasadded 1,3 propanesultone (492 mg, 4 mmole) with stirring. The reactionwas heated to 120° C. for 1.5 hr. After cooling down the reaction wasdiluted with 10 mL 0.1 N TEAB, filtered, and purified by reverse phaseHPLC eluted with a gradient of CH₃CN over 0.1 N TEAB buffer. The yieldof the product was 220 mg.

2.19 Synthesis of 2w

2u (200 mg, 0.30 mmole) dissolves in 1 mL of sulfolane at 80° C. wasadded 6-bromohexanoic acid (197 mg, 1.0 mmole) with stirring. Thereaction was heated to 130° C. for 3 hr. During which time more6-bromohexanoic acid (60 mg, 0.34 mmole) was added to the reaction.After cooling down the reaction was diluted with 10 mL of DMF. Theprecipitate was collected, washed with 1 mL DMF, EtOAc, Et₂O, driedunder vacuum. The yield of the crude product was 116 mg and was usedwithout further purification.

2.20 Synthesis of 2x

2q (37 mg, 0.10 mmole) and diphenyl formamidine (25 mg, 0.12 mmole) washeated in 4 mL of HOAc at 120° C. for 4 hr. During which time morediphenyl formamidine (12 mg, 0.06 mmole) was added to the reaction.After cooling down, the reaction was diluted with 30 mL of EtOAc, theprecipitate was collected, washed with EtOAc, Et₂O, and dried undervacuum. The yield of the crude product was 32 mg and was used withoutfurther purification.

2.21 Synthesis of 2y

To a stirring solution of 2x′ (24 mg, 0.027 mmole) and 2x (16 mg, crude)in 0.7 mL of DMF was added Et₃N (14 mg, 0.14 mmole) followed by Ac₂O (14mg, 0.14 mmole). The reaction was stirred at room temperature for 3 hrand quenched with 4 mL EtOAc. The precipitated was collected andpurified by reverse phase HPLC eluted with a gradient of CH₃CN over 0.1N TEAB buffer. The yield of the product was 6.3 mg.

2.22 Synthesis of 2y′

To a stirring solution of 2x″ (29 mg, 0.033 mmole) and 2x (16 mg, crude)in 0.7 mL of DMF was added Et₃N (16 mg, 0.16 mmole) followed by Ac₂O (16mg, 0.16 mmole). The reaction was stirred at room temperature for 3 hrand quenched with 4 mL EtOAc. The precipitated was collected andpurified by reverse phase HPLC eluted with a gradient of CH₃CN over 0.1N TEAB buffer. The yield of the product (DS374 140) was 9.6 mg.

Example 3

3.1 Synthesis of 3b, 3b′, and 3b″

3a (1.25 g, triethylammonium salt) was stirred with 5 mL of POCl₃ atroom temperature for 18 hr. POCl₃ was removed by rotavap and pumpedunder vacuum for 15 hr. The residue was dissolved in 10 mL DMF and addeddropwise to a solution of taurine (5.5 g, 44 mmole) and Na₂CO₃ (14.0 g,132 mmole) in 250 mL DMF/H₂O (1:9) over 10 min at room temperature. Thestirred for 30 min after the addition was completed. The products werepurified by reverse phase HPLC eluted with a gradient of CH₃CN over 0.1N TEAB buffer. The yield of the product 3b was 530 mg, 3b′ was 260 mg,and 3b″ was 303 mg.

3.2 Synthesis of 3c

3b (67 mg, 0.1 mmole) dissolved in 0.8 mL of sulfolane at 80° C. wasadded 1,3 propanesultone (87 mg, 0.7 mmole) with stirring. The reactionwas heated to 120° C. for 1.2 hr. After cooling down the reaction wasdiluted with 10 mL 0.1 N TEAB, filtered, and purified by reverse phaseHPLC eluted with a gradient of CH₃CN over 0.1 N TEAB buffer. The yieldof the product was 54 mg.

3.3 Synthesis of 3c′

3b (67 mg, 0.1 mmole) dissolved in 0.8 mL of sulfolane at 80° C. wasadded 6-bromohexanoic acid (196 mg, 1.0 mmole) with stirring. Thereaction was heated to 120° C. for 3 hr. After cooling down the reactionwas diluted with 10 mL 0.1 N TEAB, filtered, and purified by reversephase HPLC eluted with a gradient of CH₃CN over 0.1 N TEAB buffer. Theyield of the product was 42 mg.

3.4 Synthesis of 3d and 3d′

3c (5 mg, 0.006 mmole) and 3c′ (5 mg, 0.006 mmole) dissolved in 0.3 mLof sulfolane and 0.3 mL of pyridine at 80° C. was added 1,1,3,3tetramethoxypropane (4.0 mg, 0.025 mmole) with stirring. The reactionwas heated to 120° C. for 3 hr, during which time additional 1,1,3,3tetramethoxypropane (8.2 mg, 0.05 mmole) was added to the reaction.After cooling down the reaction was diluted with 10 mL 0.1 N TEAB,filtered, and purified by reverse phase HPLC eluted with a gradient ofCH+CN over 0.1 N TEAB buffer. The yield of the product (3d′) was 1.7 mg.Also isolated from the reaction was 3d (2 2.4 mg).

3.5 Synthesis of 3e and 3e′

Similar to the synthesis of 3d and 3d′; starting from 3b″, 3e and 3e′were prepared in 3 steps with similar yields.

3.6 Synthesis of 3f and 3f

Similar to the synthesis of 3d and 3d′; starting from 3b′, 3f and 3f′were prepared in 3 steps with similar yields.

Example 4

4.1 Synthesis of 4b

Argon was bubbled through a solution of 4a (1.0 g, 4.5 mmole),4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.72 g, 13.5 mmole), andtriethylamine (1.5 g, 14.6 mmole) in 10 mL of dioxane for 30 min,Pd(PPh₃)₂Cl₂ (160 mg, 0.23 mmole) was added to the mixture afterbubbling for 15 min. The result mixture was heated at 80° C. for 8 hrunder a slight positive pressure of argon. The solvents were removed invacuo, the residue was dissolved in DCM and purified by silica gel flashcolumn chromatography eluted with a gradient of MeOH over DCM. The yieldof the desired product was 646 mg.

4.2 Synthesis of 4d

A solution of Na₂CO₃ (240 mg, 2.26 mmole) in 2.5 mL H₂O was addeddropwise to a solution of 4c (207 mg, 0.45 mmole) and 4b (115 mg, 0.45mmole) in 5 mL of DMF with stirring. Argon was bubbled through theresult mixture for 1 hr, Pd(PPh₃)₄ (17 mg, 0.015 mmole) was added to themixture after bubbling for 30 min. The result mixture was heated at 100°C. for 6 hr under a slight positive pressure of argon. The solvents wereremoved in vacuo, the residue was redissolved in 10 mL 0.1 N TEABbuffer, filtered and purified by reverse phase HPLC eluted with agradient of CH₃CN over 0.1 N TEAB buffer. The yield of the desiredproduct was 147 mg.

4.3 Synthesis of 4e

A solution NaNO₂ (21 mg, 0.30 mmole) in 0.5 mL H₂O was added to asolution of 4d (147 mg, 0.20 mmole) in 6 mL trifluoroacetic acid and 1mL 6 N HCl at −5˜−10° C. over 2 min. During the course of addition thetemperature was kept below −5° C. The reaction was stirred for another30 min after the addition was completed. SnCl₂.2H₂O (145 mg, 0.64 mmole)in 0.5 mL 6N HCl, pre-cooled at −5° C., was added dropwise to thereaction at −5° C. over 2 min. The reaction was stirred for 4 hr as thecooling bath gradually warmed to room temperature. The reaction wasdiluted with 10 mL of H₂O, filtered, neutralized with 1 M NaOH to pH 9,and purified by reverse phase HPLC eluted with a gradient of CH₃CN over0.1 N TEAB buffer. The yield of the product was 88 mg.

4.4 Synthesis of 4f

A solution of 4e (88 mg, 0.12 mmole) and methyl isopropyl ketone (104mg, 1.2 mmole) in 3 mL HOAc was heated at 100° C. for 3 hr. HOAc wasremoved in vacuo, the residue redissolved in 0.1 N TEAB, filtered, andpurified by reverse phase HPLC eluted with a gradient of CH3CN over 0.1N TEAB buffer. The yield of the product was 77 mg.

4.5 Synthesis of 4g and 4g′

Fuming sulfuric acid (1 mL) was added to 4f (70 mg, 0.088 mmole) and thereaction stirred at room temperature for 1 hr. The reaction was quenchedwith 10 mL EtOAc pro-cooled at 4° C. The precipitate was collected bycentrifugation redissolved in 0.1 N TEAB, filtered, and purified byreverse phase HPLC eluted with a gradient of CH₃CN over 0.1 N TEABbuffer. The yield of the product, 4g was 14 mg and 4g′ was 45 mg.

4.6 Synthesis of 4h′

4g′ (21 mg, 0.021 mmole) dissolved in 0.5 mL of sulfolane at 80° C. wasadded 6-bromohexanoic acid (60 mg, 0.30 mmole) with stirring. Thereaction was heated to 130° C. for 3 hr. After cooling down the reactionwas diluted with 5 mL 0.1 N TEAB, filtered, and purified by reversephase HPLC eluted with a gradient of CH₃CN over 0.1 N TEAB buffer. Theyield of the product was 10.1 mg.

4.7 Synthesis of 4h

4g′ (24 mg, 0.034 mmole) dissolved in 0.5 mL of sulfolane at 80° C. wasadded 1,3 propanesultone (47 mg, 0.34 mmole) with stirring. The reactionwas heated to 120° C. for 1 hr. After cooling down the reaction wasdiluted with 10 mL 0.1 N TEAB, filtered, and purified by reverse phaseHPLC eluted with a gradient of CH3CN over 0.1 N TEAB buffer. The yieldof the product was 17.3 mg.

4.8 Synthesis of 4i′

4h (8.7 mg, 0.007 mmole) and 3-anilinoacrolein anil (6 mg, 0.023 mmole)was heated in 0.2 mL of HOAc and 0.2 mL of Ac₂O at 80° C. for 1 hr.After cooling down, the reaction was diluted with 4 mL of EtOAc, theprecipitate was collected, washed with EtOAc, Et₂O, and dried undervacuum. The yield of the crude product was 11.4 mg and was used withoutfurther purification.

4.9 Synthesis of 4i

4h (8.7 mg, 0.007 mmole) and diphenyl formamidine (4.7 mg, 0.023 mmole)was heated in 0.3 mL of HOAc at 120° C. for 6 hr. During which time morediphenyl formamidine (4.7 mg, 0.023 mmole) was added to the reaction.After cooling down, the reaction was diluted with 4 mL of EtOAc, theprecipitate was collected, washed with EtOAc, Et₂O, and dried undervacuum. The yield of the crude product was 10.5 mg and was used withoutfurther purification.

4.10 Synthesis of 4j

To a stirring solution of 4h′ (3.3 mg, 2.8 umole) and 4i′ (11.4 mg,crude) in 0.2 mL of DMF was added Et₃N (10 mg, 0.1 mmole) followed byAc₂O (10 mg, 0.1 mmole). The reaction was stirred at room temperaturefor 3 hr and diluted with 5 mL 0.1 N TEAB buffer. The solution wasfiltered and the product purified by reverse phase HPLC eluted with agradient of CH₃CN over 0.1 N TEAB buffer. The product was furtherpurified by ion exchange column eluted with a gradient of 1.5 N TEABbuffer with 20% ACN over 0.05 N TEAB buffer with 20% ACN. The yield ofthe product was 1.6 mg.

4.11 Synthesis of 4k

To a stirring solution of 4h′ (3.3 mg, 2.8 umole) and 4i (10.5 mg,crude) in 0.2 mL of DMF was added Et₃N (10 mg, 0.1 mmole) followed byAc₂O (10 mg, 0.1 mmole). The reaction was stirred at room temperaturefor 3 hr and diluted with 5 mL 0.1 N TEAB buffer. The solution wasfiltered and the product purified by reverse phase HPLC eluted with agradient of CH₃CN over 0.1 N TEAB buffer. The product was furtherpurified by ion exchange column eluted with a gradient of 1.5 N TEABbuffer with 20% ACN over 0.05 N TEAB buffer with 20% ACN. The yield ofthe product was 1.0 mg.

Example 5

5.1 Synthesis of 5a

To a solution of 71 mg of 2,3,3-Trimethyl-4,6-dicarboxy-3H-indole in 3.5mL of DMF, 0.21 mL of triethylamine was added followed by 0.26 g ofN,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate. Theresulting mixture was stirred at room temperature for 15 minutes togenerate the bis-N-succinimidyl ester. In a separate flask, a solutionof taurine was prepared by dissolving 0.35 g of taurine in 3.5 mL of 1 MNaHCO₃. The bis-N-succinimidyl ester solution was added in a rapiddropwise fashion into this aqueous taurine solution and after two hoursof stirring at room temperature, all the volatile components wereremoved under reduced pressure and the residual was stirred in 10 mL ofwater, filtered to remove any insoluble material and the crude waspurified on a RP C-18 column to yield 80 mg of product as itstriethylammonium salt.

5.2 Synthesis of 5b

A mixture of about 0.1 mmole of 2v as its bis-triethylammonium salt and20 mg of diphenylforamidine was heated in 3 mL of HOAc at 120 C. for 3hours. Volatile components were evaporated under reduced pressure andthe residue was dissolved in 1 mL of DMF followed by the addition of 5mL of ethyl acetate. The supernatant was decanted and the material wasused without further purification.

5.3 Synthesis of 5c

A mixture of about 0.1 mmole of 2v as its bis-triethylammonium salt, 26mg of malonaldehyde dianil hydrochloride, 1 mL of acetic anhydride washeated in 3 mL of HOAc at 120° C. for 4 hours. Volatile components wereevaporated under reduced pressure and the residue was dissolved in 1 mLof DMF followed by the addition of 5 mL of ethyl acetate. Thesupernatant was decanted and the material was used without furtherpurification.

5.4 Synthesis of 2,3,3-trimethyl-5-carboxy-3H-indole (5d)

A solution of 4-hydrazinobenzoic acid (10.0 g, 65.7 mmol),isopropylmethylketone (21.1 mL, 197 mmol) in acetic acid (35 mL) washeated under reflux in an oil bath for 20 h. After cooling to ambienttemperature the solvent was evaporated off under reduced pressure and toit was added a saturated aqueous solution of NaHCO₃ (50 mL) and washedwith CH₂Cl₂ (3×40 mL). The pH of the aqueous solution was adjusted with1 M aqueous HCl to ca. 2, and then extracted with CH₂Cl₂ (3×50 mL). Thecombined organic solution was then dried with Na₂SO₄, filtered andconcentrated to dryness under reduced pressure to yield the desiredproduct (10.7 g, 80%) as a brownish solid.

5.5 Synthesis of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-carboxylate (5e)

A solution of 2,3,3-trimethyl-5-carboxy-3H-indole (193 mg, 0.950 mmol)in 1,3-propanesultone (1 mL) was heated to 145° C. in a sealed tube for20 h. Cooled the tube to ambient temperature and to it was added ethylacetate (20 mL), stirred and the organic solvent was decanted. Repeatedthe process two more times with ethyl acetate (2×20 mL) and the oilyproduct was dried under reduced pressure. Added 6 M HCl (10 mL) to thetube and heated to 60° C. for 4 h. After cooling to ambient temperaturethe solvent was evaporated off under reduced pressure. The crude productwas then purified by reverse-phase HPLC (acetonitrile/0.1 M TEABgradient) to give 300 mg of a solid product (97% yield) afterevaporation of solvent.

5.6 Synthesis of 1-carboxypentyl-2,3,3-trimethylindoleninium-5-sulfonate(5f)

To an oven dried 250-mL round bottom flask equipped with a stir bar,condenser, and an argon balloon in an oil bath was added2,3,3-trimethylindolenine-5-sulfonate (10 g, 36.05 mmol), bromohexanoicacid (8.78 g, 45.0 mmol) and 1,2-dichlorobenzene (100 mL). Heated theoil bath to 110° C. for 24 h. Monitored the reaction with TLC (2:1CH₂Cl₂:MeOH) for the disappearance of starting material (R_(f)=0.69) andthe formation of product (R_(f)=0.22). Removed oil bath and cooled themixture to room temperature. Decanted the solvent and triturated thesolid with iPrOH (100 mL). Collected the solid using filtration funnel.Re-dissolved the solid in MeOH (300 mL) and added iPrOH (700 mL) toprecipitate the solid. The resultant solid was collected, washed withiPrOH (2×50 mL), EtOAc (2×50 mL), ether (2×50 mL) and air dried. Thesolid was placed in an amber bottle and dried in a desiccator under highvacuum overnight. There was obtained a total of 8.02 g (62%) of product.

5.7 Synthesis of 5g

A mixture of 0.71 g of1-carboxypentyl-2,3,3-trimethylindoleninium-5-sulfonate, 0.43 g ofdiphenylforamidine, and 30 mg of potassium acetate was heated at 110° C.in 5 mL of acetic anhydride for 3 hours. After cooling down to roomtemperature, 20 mL of ethyl acetate was added and the product wasobtained by filtration.

5.8 Synthesis of 5h

A mixture of 25 mg of1-(3-sulfopropyl)-2,3,3-trimethylindoleninium-5-sulfonate potassium and19 mg of diphenylforamidine was heated in about 1 mL of HOAc at 115 C.for 3 h. Another 40 mg of diphenylforamidine was added and heatingcontinued for another 4 hours. Volative components were evaporated underreduced pressure and the residue was dissolved in 1 mL of DMF and 3 mLof ethyl acetate was then added and stirred for 1 hour. The product wasobtained by filtration and used without further purification.

5.9 Synthesis of 5i

A mixture of 25 mg of1-(3-sulfoproyl)-2,3,3-trimethylindoleninium-5-sulfonate potassium and21 mg of malonaldehyde dianil hydrochloride, and 20 uL of aceticanhydride was heated in 1 mL of HOAc at 120 C for 4 hours. Volatilecomponents were removed under reduced pressure and the residue wasdissolved in 1.5 mL of DMF followed by the addition of 6 mL of ethylacetate. The supernatant was decanted and the product was used withoutfurther purification.

5.10 Synthesis of2-[(1E)-2-anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5-carboxylate(5j)

A mixture of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-carboxylate (108 mg,0.27 mmol) and N,N′-diphenylformamidine (66 mg, 0.34 mmol) in aceticacid (5 mL) was heated to reflux for 18 h. The progress of the reactionwas monitored with analytical HPLC for the disappearance of startingmaterial, and the formation of product. Extended heating may be neededto completely consume the starting material, however, some of thesymmetrical dye can also be produced. Acetic acid was removed underreduced pressure and the residual dark solid was washed with ethylacetate (3×20 mL). The dried solid was used without further purificationin the next reaction for carbocyanine dye synthesis.

5.11 Synthesis of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5-carboxylate(5k)

A mixture of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-carboxylate (300 mg,0.922 mmol) and malonaldehyde dianil hydrochloride (310 mg, 1.20 mmol)in acetic acid (10 mL) was heated to reflux for 16 h. The progress ofthe reaction was monitored with analytical HPLC for the disappearance ofthe starting material, and the formation of the product. Acetic acid wasremoved under reduced pressure and the residual dark solid was washedwith ethyl acetate (3×20 mL). The dried solid was used without furtherpurification in the next reaction for dicarbocyanine dye synthesis.

5.12 2,3,3-Trimethyl-4-carboxy-3H-indole (5l)

A solution of 3-hydrazinobenzoic acid (30.0 g, 197 mmol),isopropylmethylketone (31.7 mL, 296 mmol) in ethanol (500 mL) andsulfuric acid (5 mL) was heated under reflux in an oil bath for 20 h.After cooling to ambient temperature the solvent was evaporated offunder reduced pressure to a small volume of ˜200 mL. Collected the solidwith a filter funnel, washed with iPrOH (3×30 mL) and ethyl ether (3×30mL) and dried. Further drying of the solid in an oven at 45° C. underhigh vacuum for 18 h provided 32.59 g (81.3%) of the product.

5.13 Synthesis of 5m

The compound was obtained by following the procedure for SY360-80 using2,3,3-trimethyl-4-carboxy-3H-indole as the starting material. It wasisolated by HPLC as its triethylammonium salt.

5.14 Synthesis of 5n

The compound was prepared by following the procedure for 2v with 5m asthe starting material.

5.15 Synthesis of 5o

A mixture of 4.06 g of 2,3,3-Trimethyl-4-carboxy-3H-indole and 12.4 mLof propanesultone in 20 mL of sulfolane was heated at 130° C. for 2hours. After the reaction mixture was cooled to below 50° C., 40 mL ofethyl acetate was added while stirring was continued for another 30minutes. The solid was filtered, pumped dried and heated in 100 mL of 6N HCl at 90° C. for about 12 hours. The aqueous layer was evaporatedunder reduced pressure and the residue was stirred in 20 mL of MeOH andfiltered to obtain 3.75 g of product.

5.16 Synthesis of 5p

A mixture of 0.73 g of SY360-86 and 0.88 g of diphenylforamidine in 10mL of HOAc was heated at 120° C. for 3 hours. The acetic acid wasevaporated and the residue was dissolved in 5 mL of DMF and 30 mL ofethyl acetate was added. The supernatant was decanted and the crude thusobtained was purified on a silica gel column with water and acetonitrileto yield 90 mg of pure product.

5.17 Synthesis of 5qA and 5qB

A mixture of 0.98 g of e and 1.04 g ofN-(3-(phenylamino)-2-propenylidene)aniline hydrochloride, 1.5 mL ofacetic anhydride in 12 mL of HOAc was heated at 120° C. for 2 hours. Atwhich time another 0.5 g of N-(3-(phenylamino)-2-propenylidene)anilinehydrochloride and 1 mL of acetic anhydride were added and heatingcontinued for another 2 hours. HOAc was evaporated under reducedpressure and the residue was dissolved in 15 mL of DMF and to thissolution 105 mL of ethyl acetate was added and the crude product wasobtained by filtration. The crude product was further purified on asilica gel column with water and acetonitrile to yield 0.48 g of productwhich contained both A and B.

5.18 Synthesis of 4-methyl-5-oxo-1-hexanesulfonate (5r)

To a solution of ethyl 2-methylacetoacetate (30.0 mL, 208 mmol) wasadded 1M t-BuOK in t-BuOH (229 mL, 229 mmol) and 1,3-propanesultone(20.2 mL, 229 mmol) and heated in an oil bath to reflux for 24 h. Aftercooling to ambient temperature a solid was formed which was trituratedwith EtOAc (200 mL) and the resultant solid was collected throughfiltration, washed with ethyl acetate (2×20 mL) and dried. To the driedsolid was added 50% HCl (100 mL) and the solution was heated at 110° C.for 24 h. Solvent was evaporated off under reduced pressure andco-evaporated with acetonitrile (2×30 mL) to give 38.3 g (95%) of anoily product. The crude residue was used without further purification.

5.19 Synthesis of 5s

A mixture of 0.464 g of 3,5-dicarboxyphenylhydrazine and 0.6 g of4-Methyl-5-oxo-1-hexanesulfonate in 5 mL of HOAc was heated at 120° C.for 20 hours. Solvent was then removed under reduced pressure andreplaced by 20 mL of acetonitrile and the resulting mixture was heatedat 70° C. for another 20 hours and filtered to obtain 0.92 g of product.

5.20 Synthesis of 5t

To a solution of 0.71 g of 5s in 20 mL of DMF, 1 mL of triethylamine and1.2 g of N,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uroniumtetrafluoroborate was added and stirred at room temperature for 30minutes. In a separate container, a solution of taurine was prepared bydissolving 2.5 g of taurine in 30 mL of 1 M NaHCO₃. To this aqueoussolution of taurine, the aforementioned DMF solution was added in arapid dropwise fashion. Upon completion of addition, the resultingmixture was stirred for 1 hour at room temperature. All the volatilecomponents were evaporated under reduced pressure and the residue wasstirred in 70 mL of methanol at 50° C. for about 6 hours. The methanoliclayer was filtered and purified by a silica gel column eluting withwater and acetonitrile to yield about 0.71 g of a mixture of a mono andbis taurinated product. The mixture of products thus obtained was putthrough the same process again to improve the yield of the desiredbis-taurinated product. After the second silica gel column, thefractions containing the product were pooled and the solid was stirredin about 30 mL of methanol at 35° C. for 4 days and filtered to yieldthe desired product.

5.21 Synthesis of 5u

A mixture of 57 mg of 5t and 122 mg of propanesultone in 1 mL ofsulfolane was heated at 130° C. for 3 hours. At the end of the heating,4 mL of ethyl acetate was added and the supernatant was decanted and theresidue was pumped dried and dissolved in 3 mL of 1 N HCl and heated at70° C. for 4 hours. The aqueous layer was evaporated under reducedpressure to obtain the product and used without further purification.

5.22 Synthesis of 5v

A mixture of 142 mg of1-(3-sulfonatopropyl)-2,3,3-trimethylindoleninium-5-carboxylate, 0.37 gof 3,4-diethoxy-3-cyclobutene-1,2-dione, 0.21 mL of triethylamine in 10mL of ethanol was heated at 100 C. in a sealed container for 2 hours.Another 0.15 g of 3,4-diethoxy-3-cyclobutene-1,2-dione was added andheated for 30 additional minutes before 10 mL of water and 1 mL oftriethylamine were added and heated for another 6 hours. All volatilecomponents were removed under pressure and the residue was stirred inethyl acetate to yield a brown solid. The crude thus obtained waspurified by HPLC to yield 95 mg of the product.

5.23 Synthesis of 5wA and 5wB

A mixture of 1.38 g of 3-methoxyphenylhydrazine and 3.2 mL of3-methyl-2-butanone was heated in 5 mL of HOAc at 120 C for 3 hour.Volatile components were removed under reduced pressure and the residuewas partitioned between water and ethyl acetate. The organic layer wasdried over magnesium sulfate and purified on silica gel column elutingwith ethyl acetate/hexanes to yield 1 g of A and 0.6 g of B.

5.24 Synthesis of 5x

The compound was prepared by following the procedure for SY360-80 exceptthat the taurine was replaced with glycine and purified on silica gelcolumn.

5.25 Synthesis of 5y

The compound was prepared by following the procedure for 5a and purifiedby HPLC.

5.26 Synthesis of 5z

The compound was prepared by following the procedure for 2v using 5y asthe starting material.

5.27 Synthesis of 5aa

A mixture of 152 mg of 5w (A) and 0.49 g of propanesultone was heated in2 mL of dichlorobenzene at 120° C. for 1 hour. At the end of theheating, 10 mL of ethyl acetate was added and heated at 60° C. for 30minutes and the supernatant was decanted. Part of the crude material wasthen stirred with about 3 equivalents of 5p in DMF in the presence ofabout 5-10 equivalents of both acetic anhydride and triethylamine toyield the desired product.

5.28 Synthesis of 5ab

To a mixture of 140 mg of 2v, 94 mg of 5p in 2 mL of DMF, 104 uL oftriethylamine was added followed by 55 uL of acetic anhydride. Afterstirring at room temperature for 1.5 hours, 8 mL of ethyl acetate wasadded and the supernatant was decanted and the crude residue was pumpeddried and purified by HPLC to yield 33 mg of the desired product.

5.30 Synthesis of 5ac

The compound was prepared by mixing equal equivalents of 5z and 5p inDMF in the presence of excess 3 equivalents of acetic anhydride and 5equivalents of triethylamine. The product was precipitated out by theaddition of ethyl acetate and the crude was purified by HPLC.

5.31 Synthesis of 5ad

To a mixture of 0.1 mmole of 5b and 0.12 mmole of 2w in 2 mL of DMF atroom temperature, 0.15 mL of triethylamine and 60 uL of acetic anhydridewere added. After stirring at room temperature overnight, about 10 mL ofethyl acetate was added and the supernatant was decanted and the crudewas purified by preparation HPLC.

5.32 Synthesis of 5ae

A mixture of about 0.06 mmole of 5h and 0.1 mmole of 5w was stirred with75 uL of acetic anhydride and 0.15 mL of triethylamine in 4 mL of DMF atroom temperature overnight. Ethyl acetate was added to precipitate thecrude product which was then purified by HPLC.

5.33 Synthesis of 5af

The compound was prepared by mixing 2v and 5g in DMF in the presence oftriethylamine and acetic anhydride and purified by HPLC.

5.34 Synthesis of 5ag

To a mixture of about 100 mg of 2v and 57 mg of2-[(1E)-2-Anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5-carboxylatein 1.5 mL of DMF, 0.25 mL of triethylamine and 0.1 mL of aceticanhydride were added and stirred at room temperature overnight before 6mL of ethyl acetate was introduced. The supernatant was removed by adisposable pipette and the residue was pumped dried and purified by HPLCto yield 6.8 mg of product.

5.35 Synthesis of 5ah

The compound was prepared by following the procedure for 5an with 5g asthe starting material.

5.36 Synthesis of 5ai

A mixture of about 0.5 mmole of 5n, 0.5 mmole of2-[(1E)-2-anilinoethenyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5-carboxylate,0.1 mL of acetic anhydride and 0.35 mL of triethylamine in 3 mL of DMFwas stirred at 35 C for 8 hours. Ethyl acetate (9 mL) was added andafter stirring for 30 minutes, the crude product was filtered and wasfurther purified by HPLC to yield 23 mg of the product.

5.37 Synthesis of 5aj

The compound was prepared by following the procedure for 5an with 2ai asthe starting material.

5.38 Synthesis of 5ak

A mixture of about 0.5 mmole of 5n, 0.16 g of 5q, 0.1 mL of aceticanhydride and 0.35 mL of triethylamine in 3 mL of DMF was stirred at 35C for 8 hours. Ethyl acetate (9 mL) was added and after stirring for 30minutes, the crude product was filtered and further purified on HPLC toyield 64 mg of product.

5.39 Synthesis of 5al

A mixture of about 0.12 mmole of 2w, 25 mg of squaric acid, and 0.5 mLof triethyl orthoacetate in 4 mL of ethanol was heated at 110° C. in asealed container for 40 minutes. All volatile components were removedunder reduced pressure and the residue was dissolved in 9 mL of waterand to which 1 mL of triethylamine was added and the resulting mixturewas stirred at room temperature overnight. The aqueous layer wasevaporated and the residue stirred in 2 mL of DMF for hour before 8 mLof ethyl acetate was added and stirred for another hour. The crude thusobtained was purified on HPLC to yield 19 mg of the desired product.

5.40 Synthesis of 5am

A mixture of about 45 mg of 2v, 20 mg of 5v, and 0.5 mL of triethylorthoacetate in 8 mL of EtOH was heated in a sealed container at 100° C.for 2 hours. Another 22 mg of 5v was added and heated for another hour.All volatile components were removed under reduced pressure and thecrude was purified by HPLC to yield 3 mg of product.

5.41 Synthesis of 5an

A solution of 3 mg of the NHS ester of 5am in 100 uL of DMF was added toan aqueous solution of 15 mg 4-aminobutyric acid in 400 uL of water inthe presence of 40 uL of triethylamine. The mixture was stirred at roomtemperature for 1 h and all the volatile components were evaporatedunder reduced pressure and the crude was purified on HPLC to yield 0.5mg of the desired product.

5.42 Synthesis of 5ap

A mixture of 150 mg of 5e and 50 uL of 1,1,3,3-tetramethoxypropane in0.5 mL of pyridine was heated at reflux for 8 hours. At the end of theheating, 1.5 mL of ethyl acetate was added and the supernatant wasdiscarded. The residue was precipitate 2× by first dissolving in 1 mL ofDMF followed by the addition of 3 mL of ethyl acetate to yield 83 mg ofthe product.

5.43 Synthesis of 5ar

A mixture of 0.23 g of 5q, 0.5 mmole of 2v, 0.15 mL of acetic anhydride,and 0.42 mL of triethylamine in 10 mL of DMF was stirred at roomtemperature for 1 hour and 30 mL of ethyl acetate was added andfiltered. The crude material was purified on HPLC to yield 288 mg ofproduct.

5.44 Synthesis of 5as

The compound was prepared by following the procedure for 5an with 5aq asthe starting material

5.45 Synthesis of 5at

To 0.1 mmole of 5b in 2 mL of DMF, a solution of 0.06 mmole of 2w in 1ml of DMF was added followed by 60 uL of acetic anhydride and 0.15 mL oftriethylamine. The resulting mixture was stirred at 35° C. overnight. Atthe end of the period, 15 mL of ethyl acetate was added and filtered andthe crude was purified on preparative HPLC to obtain the desiredproduct.

5.46 Synthesis of 5au

A mixture of 0.1 mmole of 2w and 0.08 mmole of 5i was stirred in about 4mLof DMF in the presence of 75 uL of acetic anhydride and 0.15 mL oftriethylamine. Volume of solvent was reduced to about 1 mL and about 4mL of ethyl acetate was added and the supernatant was decanted and thecrude was purified on HPLC.

5.47 Synthesis of 5ay

A mixture of about 0.3 mmole of2-[(1E,3E)-4-anilinobuta-1,3-dienyl]-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indoleninium-5-carboxylate,0.15 mmole of 2v, 0.1 mL of acetic anhydride, and 0.14 mL oftriethylamine in 3 mL of DMF was stirred at room temperature for 6 hour.Ethyl acetate (14 mL) was added and the crude solid was filtered andpurified on HPLC to yield 20 mg of product.

5.48 Synthesis of 5aw

The compound was prepared by following the procedure for 5an with 5avand glycine, instead of 4-aminobutyric acid, as the starting materials.

5.49 Synthesis of 5ax

The compound was prepared by following the procedure for 5an with 5av asthe starting material

5.40 Synthesis of 5ay

The intermediate 5u obtained from 57 mg of 5t was dissolved in 1 mL ofDMF with 30 mg of 5q followed by the addition of 0.28 mL oftriethylamine, 150 uL acetic anhydride. After stirring at roomtemperature for 3 days, 6 mL of ethyl acetate was added and filtered andthe crude was purified on HPLC to yield 5.3 mg of product.

Example 6

5.1 Synthesis of 6b

A solution NaNO₂ (4.4 g, 63.7 mmole) in 15 mL H₂O was added to asuspension of 6a (13.8 g, 62.2 mmole) in 80 mL of 4 N HCl and 40 mL ofEtOH at −5˜−10° C. over 10 min. During the course of addition thetemperature was kept below −5° C. The reaction was stirred for another45 min after the addition was completed. SnCl₂.2H₂O (22.6 g, 100 mmole)in 20 mL 6N HCl, pre-cooled at −5° C., was added dropwise to thereaction at −5° C. over 10 min. The reaction was stirred for 3 hr at −5°C.˜5° C. cooling bath. The product was collected by filtration, washedwith cold 1 N HCl. The yield of the product (6b) was 13.2 g (77% as HClsalt) and was used without further purification.

5.2 Synthesis of 6c

A suspension of 6b (13.2 g, 48.0 mmole) and methyl isopropyl ketone(13.6 g, 144 mmole) in 70 mL HOAc was heated at 100° C. for 1.4 hr. HOAcwas removed in vacuo, the residue was purified by silica gel flashchromatography eluted with Hexane/EtOAc (98:2 to 90:10 gradient).Fractions containing the desired product were combined to yield 11.5 g(83%) of a brown solid.

5.3 Synthesis of 6d

A solution of 6c (7.0 g, 24.2 mmole),4,4,5,5-tetramethyl-1,3,2-dioxaborolane (8.4 g, 65.6 mmole), andtriethylamine (9.7 g, 96 mmole) in 30 mL of dioxane was bubbling withargon for 15 min. Pd(PPh₃)₂Cl₂ (1.0 g, 1.4 mmole) was added to thesolution and argon bubbling continued for another 15 min. The resultmixture was heated at 80° C. for 2 hr under a slight positive pressureof argon. The reaction was cooled down to room temp and the solvent wasremoved in vacuo. The residue was dissolved in 100 mL DCM, washed withwater (50 mL×2), brine (30 mL), dried (Na₂SO₄), and purified by silicagel flash column chromatography eluted with Hexane/EtOAc (98:2 to 90:10gradient). Fractions containing the desired product were combined toyield 6.2 g (77%) of a brown solid.

5.4 Synthesis of 6f

A solution of Na₂CO₃ (1.06 g, 10.0 mmole) in 8 mL H₂O was added dropwiseto a solution of 6d (371 mg, 2.0 mmole) and 6e (1.33 g, 2 mmole, as Et₃Nsalt) in 10 mL of DMF with stirring. Argon was bubbled through theresult mixture for 20 min. Pd(PPh₃)₄ (116 mg, 0.1 mmole) was added tothe mixture and argon bubbled for another 20 min. The result mixture washeated at 100° C. for 3 hr under a slight positive pressure of argon.The solvents were removed in vacuo, the residue was redissolved in 30 mL0.1 N TEAB buffer, filtered and purified by reverse phase HPLC elutedwith a gradient of CH₃CN over 0.1 N TEAB buffer. The yield of thedesired product, 6f, was 1.00 g (63%).

5.5 Synthesis of 6h

Similar to the synthesis of 6f, reaction of 6d and 6g yielded 60% of 6h.

5.6 Synthesis of 6i

A solution of 6f (118 mg, 0.15 mmole) and 1,3-propanesultone (112 mg,0.92 mmole) in 0.3 mL sulfolane was heated at 180° C. for 15 min. Aftercooling down the reaction was diluted with 10 mL 0.1 N TEAB, filtered,and purified by reverse phase HPLC eluted with a gradient of CH₃CN over0.1 N TEAB buffer. The yield of the product, 6i, was 65 mg (47%); alsoobtained was 58 mg (49%) of unreacted 6f.

5.7 Synthesis of 6j

Similar to the synthesis of 6i, reaction of 6h and 1,3-propanesultoneyielded 45% of 6j; also obtained was 47% of unreacted 6i.

5.8 Synthesis of 6k, 6l and 6m

A solution of 6i (21 mg, 0.023 mmole), 6j (18 mg, 0.020 mmole), ethylformate (7.4 mg, 0.1 mmole), KOAc (20 mg, 0.2 mmole) in 150 uL H₂O and600 uL MeOH was heated at 80° C. for 6 hr. The reaction was diluted with5 mL water and purified by ion exchange chromatography eluted withincreasing gradient of 1.5 M TEAB buffer/ACN (4:1) over 0.05 M TEABbuffer/ACN (4:1). Fraction containing dyes were combined and furtherpurified by reverse phase HPLC eluted with a gradient of CH₃CN over 0.1N TEAB buffer. The yields of the products were 6k: 2.7 mg (7%), 6l: 4.5mg (12%), and 6m: 1.1 mg (3%).

5.9 Synthesis of 6n

Fuming sulfuric acid (100 uL) was added to 61 (2.5 mg, 1.3 umole), thereaction was agitated for 12 min at 25° C. The reaction was diluted with1 mL EtOAc and 1 mL Et₂O, the precipitate was collected bycentrifugation and purified by reverse phase HPLC eluted with a gradientof CH₃CN over 0.1 N TEAB buffer. The yield of the product, 6n, was 2.6mg (88%).

The present invention provides, inter alia, novel cyanine dyes,conjugates incorporating these dyes and method of using the dyes andconjugates. While specific examples have been provided, the abovedescription is illustrative and not restrictive. Any one or more of thefeatures of the previously described embodiments can be combined in anymanner with one or more features of any other embodiments in the presentinvention. Furthermore, many variations of the invention will becomeapparent to those skilled in the art upon review of the specification.The scope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims along with their full scope ofequivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

What is claimed is:
 1. A cyanine compound or conjugate having thestructure of Formula (I) or (II):

wherein n is an integer selected from 1, 2, and 3; a, e, and j areindependently selected from the integers 1, 2, 3, and 4, such that whena is 2 or greater, each R^(a) is independently selected, when e is 2 orgreater, each R^(e) is independently selected, and when j is 2 orgreater, each R^(j) is independently selected; b and e′ areindependently selected from the integers 0, 1, and 2, such that when bis 2, each R^(b) is independently selected, and when e′ is 2, eachR^(e′) is independently selected; R^(a), R^(b), R^(e), R^(e′), andR^(j), are independently selected from C(O)R⁹, OR¹², NR¹²R¹³,CR¹²C(O)R¹³, NR¹²C(O)₂R¹³, SO₃H, and C(O)NR¹²R¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, and a bond toM; R^(f), R^(g), R^(h), and R^(i) are members independently selectedfrom halogen, substituted or unsubstituted alkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl andsubstituted or unsubstituted heteroalkyl, and a bond to M; R^(c), andR^(d) are independently selected from a bond to M, and alkyl substitutedwith a member selected from sulfonic acid, carboxylic acid, phosphonicacid, and phosphoric acid, and heteroalkyl substituted with a memberselected from sulfonic acid, carboxylic acid, phosphonic acid, andphosphoric acid; wherein R⁹ is a member selected from OR¹⁰, and a bondto M; R¹⁰ is a member selected from H and substituted or unsubstitutedalkyl; and R¹² and R¹³ are members independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,and a bond to M, wherein M comprises a linker bound to a carriermolecule.
 2. The cyanine compound or conjugate according to claim 1,wherein said linker bound to a carrier molecule is a linker bound to amoiety having the structure of Formula III:

wherein u is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8; r is 0or 1; and Y is a nucleobase.
 3. The cyanine compound or conjugateaccording to claim 1, said compound or conjugate having the structure ofFormula IV:


4. The cyanine compound or conjugate according to claim 1, said compoundor conjugate having the structure of Formula V:


5. The cyanine compound or conjugate according to claim 1, said compoundor conjugate having the structure of Formula VI:


6. The cyanine compound or conjugate of claim 1, comprising at least oneM moiety, wherein the linker of this moiety comprises the structureC(O)NH(CH₂)_(t), wherein the index t is an integer selected from 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, and
 12. 7. The cyanine compound orconjugate of claim 1, comprising at least one M moiety, wherein saidlinker of this moiety comprises a polyvalent scaffold.
 8. The cyaninecompound or conjugate according to claim 7, wherein said polyvalentscaffold is a polyvalent arylene scaffold.
 9. The cyanine compound orconjugate of claim 7, wherein the polyvalent scaffold comprises a secondcyanine moiety bound thereto, said second cyanine moiety having astructure according to Formula I or Formula II.
 10. The cyanine compoundor conjugate of claim 1, comprising at least one M, wherein the linkercomprises biotin bound to streptavidin.
 11. The cyanine compound orconjugate according to claim 1, wherein said cyanine compound orconjugate has a structure selected from the following formulae:


12. The cyanine compound according to claim 1, wherein the linker of theM moiety includes a —C(O)—.
 13. The cyanine compound or conjugateaccording to claim 2, having a structure selected from the followingformulae:


14. The cyanine compound or conjugate according to claim 1, having theformula:M-(R^(D))₂ wherein each R^(D) has a structure independently selectedfrom Formula I and Formula II.
 15. The cyanine compound or conjugateaccording to claim 7, wherein M comprises the polyvalent arylenescaffold moiety:


16. The cyanine compound or conjugate according to claim 13, having theformula:

wherein each R^(D) has a structure independently selected from Formula Iand Formula II.
 17. The cyanine compound or conjugate according to claim2, having at least one Y moiety comprising a natural nucleobase.
 18. Thecyanine compound or conjugate according to claim 1, wherein the carriermolecule comprises a member selected from a nucleic acid, a protein, aglycoprotein, a peptide, a saccharide, a hormone, a receptor, anantigen, a substrate, metabolite, a transition state analog, a cofactor,an inhibitor, a drug, a dye, a nutrient, and a growth factor.
 19. Thecyanine compound or conjugate according to claim 1, wherein the carriermolecule comprises a member selected from a solid support, a virus and amicroorganism.
 20. A method of monitoring an enzyme reaction, saidmethod comprising: (a) forming a reaction mixture by contacting saidenzyme with a cyanine compound or conjugate according to claim 1,wherein said cyanine compound or conjugate is a substrate for saidenzyme under conditions sufficient for said enzyme and said cyaninecompound or conjugate to react; and (b) monitoring fluorescence of saidreaction mixture, thereby monitoring said enzyme reaction.
 21. Themethod according to claim 20, wherein said enzyme is a DNA polymeraseand said cyanine compound or conjugate comprises a nucleic acid moiety,which is said substrate for said enzyme.
 22. The method according toclaim 20, wherein said enzyme reaction is template directed DNAsynthesis.
 23. The method according to claim 20, wherein said reactionis a component of a single molecule DNA sequencing analysis.
 24. Acyanine compound or conjugate having the structure of Formula (I) or(II):

wherein n is an integer selected from 1, 2, and 3; a is selected fromthe integers 2, 3 and 4, and each R^(a) is independently selected; e,and j are independently selected from the integers 1, 2, 3, and 4, suchthat when e is 2 or greater, each R^(e) is independently selected, andwhen j is 2 or greater, each R^(j) is independently selected; b and e′are independently selected from the integers 0, 1, and 2, such that whenb is 2, each R^(b) is independently selected, and when e′ is 2, eachR^(e′) is independently selected; R^(a), R^(b), R^(e), R^(e′), andR^(j), are independently selected from C(O)R⁹, OR¹², NR¹²R¹³,CR¹²C(O)R¹³, NR¹²C(O)₂R¹³, SO₃H, and C(O)NR¹²R¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, and a bond toM; R^(f), R^(g), R^(h), and R^(i) are members independently selectedfrom halogen, substituted or unsubstituted alkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl andsubstituted or unsubstituted heteroalkyl, and a bond to M; R^(c), andR^(d) are independently selected from a bond to M, and alkyl substitutedwith a member selected from sulfonic acid, carboxylic acid, phosphonicacid, and phosphoric acid, and heteroalkyl substituted with a memberselected from sulfonic acid, carboxylic acid, phosphonic acid, andphosphoric acid; wherein R⁹ is a member selected from OR¹⁰, and a bondto M; R¹⁰ is a member selected from H and substituted or unsubstitutedalkyl; and R¹² and R¹³ are members independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,and a bond to M, wherein M comprises a linker bound to a carriermolecule.
 25. A cyanine compound or conjugate having the structure ofFormula (I) or (II):

wherein n is an integer selected from 1, 2, and 3; a, e, and j areindependently selected from the integers 1, 2, 3, and 4, such that whena is 2 or greater, each R^(a) is independently selected, when e is 2 orgreater, each R^(e) is independently selected, and when j is 2 orgreater, each R^(j) is independently selected, and at least one ofR^(a), R^(b), R^(e) and Rj is:

in which each Z is independently selected from SO₃H and CO₂H, each x isan integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and10, and each R^(N) is independently selected from H, and substituted orunsubstituted alkyl; b and e′ are independently selected from theintegers 0, 1, and 2, such that when b is 2, each R^(b) is independentlyselected, and when e′ is 2, each R^(e′) is independently selected;R^(a), R^(b), R^(e), R^(e′), and R^(j), are independently selected fromC(O)R⁹, OR¹², NR¹²R¹³, CR¹²C(O)R¹³, NR¹²C(O)₂R¹³, SO₃H, and C(O)NR¹²R¹³,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,and a bond to M; R^(f), R^(g), R^(h), and R^(i) are membersindependently selected from halogen, substituted or unsubstituted alkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted heteroalkyl, and a bond toM; R^(c), and R^(d) are independently selected from a bond to M, andalkyl substituted with a member selected from sulfonic acid, carboxylicacid, phosphonic acid, and phosphoric acid, and heteroalkyl substitutedwith a member selected from sulfonic acid, carboxylic acid, phosphonicacid, and phosphoric acid; wherein R⁹ is a member selected from OR¹⁰,and a bond to M; R¹⁰ is a member selected from H and substituted orunsubstituted alkyl; and R¹² and R¹³ are members independently selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,and a bond to M, wherein M comprises a linker bound to a carriermolecule.